Cnp variants, conjugates thereof, and formulations

Abstract

This document provides pharmaceutical compositions comprising variants and prodrugs of the C-type natriuretic peptide (CNP) disclosed herein. It also provides methods for treating bone-related conditions or skeletal dysplasia using the disclosed compositions, and pharmaceutical kits comprising the disclosed compositions.

Description

[0001] Cross-reference of related applications

[0002] This application claims priority to U.S. Provisional Applications No. 63 / 597,246 (filed November 8, 2023); No. 63 / 623,961 (filed January 23, 2024); No. 63 / 651,801 (filed March 24, 2024); No. 63 / 660,347 (filed June 14, 2024); No. 63 / 695,491 (filed September 17, 2024); and No. 63 / 711,678 (filed October 24, 2024), each of which is incorporated herein by reference in its entirety.

[0003] Materials submitted electronically and incorporated by reference

[0004] The sequence list is part of this disclosure and is submitted along with this specification in the form of an XML file. The XML file containing the sequence list is named "70213_Seqlisting.xml", was created on November 7, 2024, and has a size of 98,392 bits. The subject of the sequence list is incorporated herein by reference in its entirety. Technical Field

[0005] This disclosure generally relates to variants of C-type natriuretic peptide (CNP), pharmaceutical compositions comprising CNP variants, and methods of use. CNP variants can be used as therapeutic agents to treat diseases that respond to CNP, including but not limited to bone-related conditions such as skeletal dysplasia (e.g., achondroplasia). Background Technology

[0006] C-type natriuretic peptide (CNP) (Biochem. Biophys. Res. Commun., 168: 863-870 (1990) (For the CNP precursor protein, i.e. NPPC, GenBank accession number NP_077720) (J. Hypertens., 10: 907-912 (1992)) is a small single-chain peptide belonging to the peptide family (ANP, BNP, CNP) with a 17-amino acid ring structure (Levin et al., N. Engl. J. Med., 339: 863-870 (1998)) and plays an important role in a variety of biological processes. CNP interacts with natriuretic peptide receptor-B (NPR-B, GC-B) to stimulate the production of cyclic guanosine monophosphate (cGMP) (J. Hypertens., 10:1111-1114) (1992)). CNP is widely expressed, including in the central nervous system, reproductive tract, bone and vascular endothelium (Hypertension, 49: 419-426 (2007)).

[0007] In humans, CNP is initially produced by the C-type natriuretic peptide precursor (NPPC) gene as a single-chain proto-peptide with 126 amino acids (Biochemistry and Biophysics Research Communications, 168: 863-870 (1990)). Removal of the signal peptide produces pro-CNP, which is further cleaved by the endopeptide furin to produce an active peptide (CNP-53) containing 53 amino acids. This active peptide is secreted and cleaved again by an unknown enzyme to produce a mature peptide (CNP-22) containing 22 amino acids (Wu, Journal of Biochemistry, 278: 25847-852 (2003)). CNP-53 and CNP-22 have different distributions. CNP-53 is mainly distributed in tissues, while CNP-22 is mainly found in plasma and cerebrospinal fluid (J. Alfonzo, *Receptor and Signal Transduction Journal*, 26: 269-297 (2006)). Both CNP-53 and CNP-22 bind to NPR-B in similar ways.

[0008] Downstream signaling mediated by cGMP production influences a variety of biological processes, including endochondral ossification. For example, knockout of CNP or NPR-B genes in mouse models results in dwarfism and shorter long bones and vertebrae. Mutations in human NPR-B that block appropriate CNP signaling have been identified, and these mutations cause dwarfism (Olney et al., *J. Clin. Endocrinol. Metab.* 91(4): 1229-1232 (2006); Bartels et al., *Am. J. Hum. Genet.* 75: 27-34 (2004)). In contrast, mice engineered to produce elevated levels of CNP exhibit elongated long bones and vertebrae.

[0009] The therapeutic use of CNP (CNP22) is limited by its short plasma half-life, which has been shown to be 2.6 minutes in humans (Journal of Clinical Endocrinology & Metabolism, 78: 1428-35 (1994)). CNP variants with longer in vivo serum half-lives and exhibiting similar or improved activity to wild-type CNP are important for sustainable therapeutic strategies. Summary of the Invention

[0010] This disclosure relates to novel variants of C-type natriuretic peptide (CNP) having increased circulating half-life and stability in aqueous media, pharmaceutical compositions comprising such CNP variants, and methods of treating conditions that respond to CNP using such CNP variants, including but not limited to bone-related conditions such as achondroplasia.

[0011] In various embodiments, this disclosure provides a C-type natriuretic peptide (CNP) variant selected from the group consisting of:

[0012] PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5);

[0013] PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1);

[0014] PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC(SEQ ID NO: 6); and

[0015] PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5).

[0016] In various embodiments, this disclosure provides a C-type natriuretic peptide (CNP) variant selected from the group consisting of:

[0017] PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5);

[0018] PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1);

[0019] PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6);

[0020] PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); and

[0021] PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7).

[0022] In various embodiments, the variant peptide further comprises an acetyl group. In various embodiments, the acetyl group is located at the N-terminus of the peptide. In various embodiments, the peptide further comprises an OH or NH2 group at the C-terminus.

[0023] In various embodiments, the variant peptide comprises a conjugate moiety. In various embodiments, the conjugate moiety is located on a residue of a CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the conjugate moiety is located on a lysine residue. In various embodiments, the conjugate moiety comprises one or more acid moieties. In various embodiments, the acid moieties are hydrophobic acids.

[0024] In various embodiments, the conjugated portion comprises one or more acidic portions linked to a hydrophilic spacer. In various embodiments, the hydrophilic spacer is any amino acid. In various embodiments, the hydrophilic spacer is γ-glutamic acid (yGlu). In various embodiments, the hydrophilic spacer is OEG (8-amino-3,6-dioxanoic acid). In various embodiments, the hydrophilic spacer is γ-glutamic acid (yGlu) or OEG (8-amino-3,6-dioxanoic acid). In various embodiments, the hydrophilic spacer is γ-glutamic acid (yGlu) linked to one or more OEG (8-amino-3,6-dioxanoic acid). In various embodiments, the acidic portion is a fatty acid. Exemplary fatty acids include short-chain, medium-chain, or long-chain fatty acids, or dicarboxylic acid fatty acids. In various embodiments, the fatty acid is saturated or unsaturated. This encompasses C-6 to C-20 fatty acids, including but not limited to C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 saturated or unsaturated fatty acids. In various embodiments, the fatty acid is decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, eicosanoic acid, or their diacids.

[0025] In various embodiments, the acid moiety and the hydrophilic spacer have the structure AEEA-AEEA-yGlu-C18DA. In various embodiments, the acid moiety and the hydrophilic spacer have the structure:

[0026] ,

[0027] in" "" indicates the connection point with the CNP variant. In various embodiments, " "" indicates a connection point with a hydrolyzable connector, which is connected to the CNP variant. In various embodiments, the hydrolyzable connector is capable of releasing the complete CNP variant.

[0028] In various embodiments, CNP variants have the following structure:

[0029] .

[0030] In various embodiments, the International Union of Pure and Applied Chemistry (IUPAC) name for the CNP variant is...

[0031] (4R,10S,16S,19S,22S,28S,31S,34S,37S,40S,43S,49S,52R)-52-(2-((S)-2-((S)-2-((S)-2-( 2-((S)-2-((S)-2-((S)-2-((S)-2-(2-((S)-2-((S)-2-((S)-2-((S)-2-(S)-2-((S)-2-((S)-1) -(L-prolyl-glycyl-L-glutamine-l-glutamine-L-histyl-pyrrolidine-2-carboxamido)-4-amino-4-oxobutamido)propamido)-5-guanidinopentamido)-6-aminohexano)-3-(4-hydroxyphenyl)propamido)-6-aminohexano)acetamido)propamido)-4-amino-4-oxobutamido)-6-aminohexano)-6-aminohexano)acetamido)- 4-Methylpentamido)-3-hydroxypropamido)-6-aminohexano)acetami)-49-benzyl-28-((S)-sec-butyl)-34-(carboxymethyl)-40-((S)-33,51-dicarboxy-8-(2-hydroxyethyl)-6,12,21,30,35-pentoxo-14,17,23,26-tetraoxa-5,8,11,20,29,34-hexaazapentadecyl)-31-(3-guanidinopropyl) -16,22-bis(hydroxymethyl)-10,37,43-triisobutyl-19-(2-(methylthio)ethyl)-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-hexadecoxo-1,2-dithia-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-hexaazacyclopentasan-4-carboxylic acid.

[0032] In various embodiments, the chemical formula of the CNP variant is C 217 H 363 N 61 O 65 S3.

[0033] In various embodiments, the exact mass of the CNP variant is 4959.61.

[0034] In various embodiments, the molecular weight of the CNP variant is 4962.83.

[0035] In various embodiments, the CNP variant having the conjugated portion is a component of the release composition. In various embodiments, the release composition is a prolonged release composition. In various embodiments, the CNP variant comprising the conjugated portion and the hydrolyzable linker is capable of releasing the CNP variant, wherein at pH 7 to 7.6, (i) less than about 20% of the CNP variant is released on day 1; and (ii) about 90% of the CNP variant is released weekly, or about 90% of the CNP variant is released every two weeks, or about 90% of the CNP variant is released monthly.

[0036] In various embodiments, (i) less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the peptide is released on day 1 at pH 7.0 to 7.6; and (ii) about 90% of the peptide is released weekly, or bi-weekly, or monthly at pH 7 to 7.6. Upon further consideration, (i) at pH 7.0 to 7.6, less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the peptides are released on day 1; and (ii) at pH 7 to 7.6, about 70%, about 80%, or about 90% of the peptides are released weekly; or about 70%, about 80%, or about 90% of the peptides are released every two weeks; or about 70%, about 80%, or about 90% of the peptides are released every three weeks; or about 70%, about 80%, or about 90% of the peptides are released monthly; or (ii) at pH At levels 7 to 7.6, approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released weekly; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released every two weeks; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released every three weeks; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released monthly.

[0037] In various embodiments, the variant has the following structure:

[0038] PGQEH PQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-YGIU-C18DA) LDRIGSMSGLGC (SEQID NO: 5), or

[0039] AC-PGQEH PQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-YGIU-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 8).

[0040] In various embodiments, the variants are selected from the group consisting of: Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 8); Ac-PGQEH PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 9); Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10); Ac-PGQEH PNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 11); Ac-PGQEH PQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 12); Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 13); and Ac- PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 14).

[0041] In various embodiments, the variants comprise one or more linker groups. In various embodiments, the linker is located on a residue of a CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the linker is located on a lysine residue.

[0042] In various embodiments, the connector is a pyrolytic or soluble connector. In some of the embodiments, the connector is a hydrolyzable connector.

[0043] In various embodiments, the CNP variant is linked to the conjugation moiety via a linker. In various embodiments, the linker is linked to the conjugation moiety via a hydrophilic spacer of the conjugation moiety. In various embodiments, the linker is aminoethoxy-2-ethoxyacetic acid (AEEA). In various embodiments, the linker is a bicin-type linker or a peptide-like linker, meaning a linker having a cleavage mechanism similar to bis-2-hydroxyethylglycine (bicin), but actually via asymmetric N-alkyl peptide (i.e., peptide-like) cleavage. In various embodiments, the linker is an electron linker based on non-enzymatic β-elimination. In various embodiments, the electron linker comprises an SO2 moiety. Examples of linkers illustrated with CNP conjugates are described in... Figure 1See also Santi et al., Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci USA) 109:6211-6216, 2012. In various embodiments, the pyrolytic or soluble joint, particularly the hydrolytic joint, pyrolytically releases the CNP variant from the fused portion. Exemplary embodiments in which this may occur are depicted in Figure 9 middle.

[0044] In various embodiments, the conjugated portion is a synthetic polymer group. In various embodiments, the variant comprises a synthetic polymer group coupled to the variant via a hydrolyzable linker. In various embodiments, the synthetic polymer group comprises a hydrophilic polymer moiety. In various embodiments, the hydrophilic polymer moiety comprises polyethylene glycol (PEG). In various embodiments, the hydrophilic polymer moiety comprises polyethylene glycol (PEG) having a chain length of 6 to 20 atoms. For the purposes of this conjugation, the synthetic polymer group is not a peptide.

[0045] In various embodiments, the variant peptides are prepared synthetically.

[0046] In various embodiments, the variant peptides were stable for 10 days at about 37°C and pH 7.0 to 7.6. In various embodiments, the variant peptides were stable for at least 10 days at about 37°C and pH 7.0 to 7.4. In various embodiments, the variant peptides were stable for at least 10 days at about 37°C and pH 7.2 to 7.6.

[0047] In various embodiments, the variant peptide is stable to deamidation. In various embodiments, the variant peptide is stable to oxidation. In various embodiments, the variant peptide is stable to deamidation and / or oxidation, or a combination thereof. In various embodiments, methionine is replaced by oroleucine. In various embodiments, almost no detectable deamidation occurs after 10 days.

[0048] In various embodiments, the variant peptide has a half-life of about 10 days at about 37°C and pH 7.0 to 7.6. In various embodiments, the variant peptide has a half-life of about 10 days at about 37°C and pH 7.0 to 7.4. In various embodiments, the variant peptide has a half-life of about 10 days at about 37°C and pH 7.2 to 7.6. In various embodiments, the variant peptide has a half-life of at least 10 days at about 37°C and pH 7.0 to 7.6. In various embodiments, the variant peptide has a half-life of at least 10 days at about 37°C and pH 7.0 to 7.4. In various embodiments, the variant peptide has a half-life of at least 10 days at about 37°C and pH 7.2 to 7.6. In various embodiments, the half-life is at least about 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 30 days or longer.

[0049] In various embodiments, the EC50 of the variant peptide is 0.1 to 10 nM in cGMP assays. In various embodiments, the EC50 of the variant peptide is 0.1 to 25 nM in cGMP assays.

[0050] In various embodiments, under physiological conditions, such as about 37°C and pH 7.0 to 7.6, after 10 days in an aqueous medium, more than 45% of the variant peptides were detected. In various embodiments, under physiological conditions, such as about 37°C and pH 7.0 to 7.6, after 10 days in an aqueous medium, more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the variant peptides were detected.

[0051] In various embodiments, more than 45% of the variant peptides were detected after 10 days in an aqueous medium at 37°C and pH 7.4. In various embodiments, more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the variant peptides were detected after 10 days in an aqueous medium at 37°C and pH 7.4.

[0052] In various embodiments, under physiological conditions, such as approximately 37°C and pH 7.0 to 7.6, after 10 days, more than 45% of the variant peptide was detected in plasma. In various embodiments, under physiological conditions, such as approximately 37°C and pH 7.0 to 7.6, after 10 days, more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the variant peptide was detected in plasma.

[0053] In various embodiments, more than 45% of the variant peptides were detected in plasma after 10 days at 37°C and pH 7.4. In various embodiments, more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the variant peptides were detected in plasma after 10 days at 37°C and pH 7.4.

[0054] In various embodiments, the variant peptide is conjugated with lipids, fatty acids, hydrophilic spacers or linkers, or optionally combinations thereof. In various embodiments, the linker is a hydrophilic polymer portion. In various embodiments, the hydrophilic polymer portion is a synthetic hydrophilic polymer portion.

[0055] In various embodiments, the variant peptide has a longer half-life compared to Pro-Gly-CNP37. In various embodiments, the variant peptide has a longer half-life compared to CNP-22. In various embodiments, the variant peptide has a longer half-life compared to Pro-Gly-CNP37 and / or CNP-22. In various embodiments, the variant peptide has a longer half-life in vitro and / or in vivo compared to Pro-Gly-CNP37 and / or CNP-22. In various embodiments, the CNP prodrug compositions exhibit lower Cmax and higher AUC compared to free drugs, such as equivalent compositions lacking acid moieties, spacers, and hydrolyzable linkers.

[0056] This disclosure further provides a pharmaceutical composition comprising the CNP variant described herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0057] In various embodiments, the composition is a lyophilized formulation prepared from a preparation comprising a citric acid / citrate buffer or an acetate / acetate buffer with a pH of about 4 to about 6. In various embodiments, the lyophilized formulation is prepared from a preparation further comprising an isotonic adjuster or build-up agent selected from the group consisting of mannitol, sucrose, sorbitol, trehalose, polysorbate 80, and combinations thereof. In various embodiments, the lyophilized formulation is prepared from a preparation further comprising an antioxidant selected from the group consisting of methionine, ascorbic acid, salts of ascorbic acid, thioglycerol, and combinations thereof. In various embodiments, the CNP variant composition is supplied as a 0.8 mg to 10 mg reconstituted lyophilized powder. In various embodiments, the CNP variant composition is supplied as a 0.8 mg or 2 mg lyophilized, preservative-free reconstituted powder.

[0058] In various embodiments, the composition is a lyophilized formulation prepared from a preparation comprising a histidine buffer (including its salts, its solvates, and solvates of its salts) or an L-histidine buffer with a pH of about 4 to about 6. In various embodiments, the lyophilized formulation is prepared from a preparation additionally comprising an isotonic adjuster or a buildup agent selected from the group consisting of mannitol, sucrose, sorbitol, trehalose, polysorbate 80, and combinations thereof. In various embodiments, the lyophilized formulation is prepared from a preparation additionally comprising an antioxidant selected from the group consisting of methionine, ascorbic acid, salts of ascorbic acid, thioglycerol, and combinations thereof. In various embodiments, the CNP variant composition is supplied as a lyophilized powder to be reconstituted, ranging from 1 mg to 300 mg. For example, in some embodiments, the CNP variant composition is supplied as a lyophilized powder in amounts of 10 to 290 mg (e.g., 25 to 250 mg, 50 to 200 mg, and 75 to 150 mg). In some embodiments, the CNP variant composition is supplied as a lyophilized powder in amounts of 10 to 50 mg (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 mg). In various embodiments, the CNP variant composition is supplied as a lyophilized, preservative-free, reconstituteable powder in amounts of 13 mg or 39 mg. This document also covers formulations comprising (a) the CNP variant peptides described herein and (b) one or more components selected from the group consisting of buffers, isotonic agents, stabilizers, and anti-adsorption agents. In a particularly preferred embodiment, the buffer used in the formulation may be L-histidine, histidine monohydrochloride monohydrate, or a combination thereof. In other preferred embodiments, the isotonic agent used in the formulation of the present invention may be trehalose dihydrate, D-mannitol, or a combination thereof. In other preferred embodiments, the stabilizer used in the formulation of the present invention is L-methionine. In other preferred embodiments, the anti-adsorbent used in the formulation of the present invention is polysorbate 80. In various embodiments, the formulation of the present invention is in lyophilized form, liquid form, or liquid form reconstituted from a pre-lyophilized form. In some embodiments, the formulation of the present invention is preservative-free and optionally may be contained in untreated type 1 borosilicate glass vials. Optionally, the pH of the formulation of the present invention is in the range of about 5.0 to about 6.0 (e.g., 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, and 6.0). In some embodiments, the pH of the disclosed formulation is about 5.5.In other embodiments, the formulations of the present invention comprise the CNP variant peptide described herein, L-histidine, histidine monohydrochloride monohydrate, trehalose dihydrate, D-mannitol, L-methionine, and polysorbate 80. In some preferred embodiments, the CNP variant peptide is present at a concentration between about 10.0 mg / ml and about 30.0 mg / ml, L-histidine is present at a concentration between about 0.18 mg / ml and about 0.50 mg / ml, histidine monohydrochloride monohydrate is present at a concentration between about 0.75 mg / ml and about 2.26 mg / ml, trehalose dihydrate is present at a concentration between about 30 mg / ml and about 70 mg / ml, D-mannitol is present at a concentration between about 10 mg / ml and about 20.0 mg / ml, L-methionine is present at a concentration between about 0.5 mg / ml and about 1.5 mg / ml, and polysorbate 80 is present at a concentration between about 0.01 mg / ml and about 0.1 mg / ml. In other embodiments, particularly preferred embodiments, the CNP variant is present at a concentration of about 10.0 mg / ml or 30 mg / ml, L-histidine is present at a concentration of about 0.347 mg / ml, histidine monohydrochloride monohydrate is present at a concentration of about 1.627 mg / ml, trehalose dihydrate is present at a concentration of about 58.00 mg / ml, D-mannitol is present at a concentration of about 15.0 mg / ml, L-methionine is present at a concentration of about 0.73 mg / ml, and polysorbate 80 is present at a concentration of about 0.05 mg / ml. In other embodiments, the CNP variant peptide is present at a concentration between about 0.4 mg / ml and about 3.5 mg / ml, citrate monohydrate is present at a concentration between about 0.15 mg / ml and about 0.40 mg / ml, sodium citrate dihydrate is present at a concentration between about 0.5 mg / ml and about 1.5 mg / ml, trehalose dihydrate is present at a concentration between about 30 mg / ml and about 70 mg / ml, D-mannitol is present at a concentration between about 10 mg / ml and about 20.0 mg / ml, L-methionine is present at a concentration between about 0.5 mg / ml and about 1.5 mg / ml, and polysorbate 80 is present at a concentration between about 0.01 mg / ml and about 0.1 mg / ml.In another embodiment, the CNP variant is present at a concentration of about 0.8 mg / ml, 2.0 mg / ml, or 5.0 mg / ml, citrate monohydrate is present at a concentration of about 0.28 mg / ml, sodium citrate dihydrate is present at a concentration of about 1.08 mg / ml, trehalose dihydrate is present at a concentration of about 58.01 mg / ml, D-mannitol is present at a concentration of about 15.0 mg / ml, L-methionine is present at a concentration of about 0.73 mg / ml, and polysorbate 80 is present at a concentration of about 0.05 mg / ml.

[0059] In various embodiments, the composition is a prolonged release composition.

[0060] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37) (BMN 111) (SEQ ID NO: 1).

[0061] A method for treating bone-related conditions or skeletal dysplasia in a subject in need is also provided, the method comprising administering to the subject a composition comprising a CNP variant described herein.

[0062] In various embodiments, bone-related disorders or skeletal dysplasia are selected from the group consisting of: osteoarthritis, hypophosphatemic rickets, achondroplasia, decreased cartilage formation, short stature, dwarfism, osteochondrodysplasia, lethal achondroplasia, osteogenesis imperfecta, chondrodysplasia, punctate chondrodysplasia, homozygous achondroplasia, camptomelic dysplasia, congenital lethal hypophosphatase disease, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, pedicled punctate chondrodysplasia, Jansen-type metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, skeletal dysplasia, malformation dysplasia, congenital short femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelic dysplasia. dysplasia), Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral bone developmental disorders, Kniest dysplasia, fibrocartilage hyperplasia, Roberts syndrome, acromegaly, microlimb syndrome, Morquio syndrome, Kniest syndrome, varicocele, and vertebral epiphyseal dysplasia.

[0063] In various embodiments, bone-related disorders or skeletal dysplasia are selected from the group consisting of: osteoarthritis, hypophosphatemic rickets, achondroplasia, decreased cartilage formation, short stature, dwarfism, osteochondrodysplasia, lethal achondroplasia, osteogenesis imperfecta, chondrodysplasia punctate chondrodysplasia, homozygous achondroplasia, brachydactyly, congenital lethal hypophosphatase disease, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, pedicled punctate chondrodysplasia, Janssen type metaphyseal dysplasia. Infertility, congenital vertebral epiphyseal dysplasia, osteogenesis imperfecta, malformation dysplasia, congenital short femur, Langer type midlimb dysplasia, Nivig type midlimb dysplasia, Robinnoc syndrome, Reinhardt syndrome, acrodysplasia, peripheral bone developmental disorders, Knifell's dysplasia, fibrocartilage hyperplasia, Roberts syndrome, acromegaly, microlimb syndrome, Moquer syndrome, Knifell's syndrome, variability dysplasia, vertebral epiphyseal metaphysplasia, and osteoporosis.

[0064] In various embodiments, CNP variants can be used as adjuncts or alternatives to growth hormone for the treatment of idiopathic short stature and other skeletal dysplasia.

[0065] In various embodiments, bone-related disorders, skeletal dysplasia, or short stature are caused by NPR2 mutations, SHOX mutations (Turner's syndrome / Leri Weill disease), or PTPN11 mutations (Noonan's syndrome).

[0066] In various embodiments, bone-related disorders, skeletal dysplasia, or short stature are caused by NPR2 mutations, SHOX mutations (Turner syndrome / Lreywell disease), PTPN1 1 mutations (Noonan syndrome), or insulin-like growth factor 1 receptor (IGF1R).

[0067] In various embodiments, CNP variants can be used to treat growth plate disorders and short stature, including familial short stature, dominant familial short stature (also known as dominant hereditary short stature), or idiopathic short stature. In various embodiments, short stature or growth plate disorders are the result of mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, or FGFR3.

[0068] In various embodiments, CNP variants can be used to treat growth plate disorders and short stature, including familial short stature, dominant familial short stature (also known as dominant hereditary short stature), or idiopathic short stature. In various embodiments, short stature or growth plate disorders are the result of mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, SHOX, NPR2, NPPC, or FGFR3.

[0069] In various embodiments, growth plate disease or short stature is associated with one or more mutations in genes related to RAS proteinopathy.

[0070] In various embodiments, bone-related disorders, skeletal dysplasia, or short stature are caused by RAS proteinopathy. In various embodiments, RAS proteinopathy is Noonan syndrome, Costello syndrome, cardiofacial skin syndrome, neurofibromatosis type 1, or leopard syndrome.

[0071] In one embodiment, RAS proteinopathy is hereditary type 1 gingival fibromatosis.

[0072] In various embodiments, CNP variants can be used to treat growth plate disorders and short stature, including familial short stature, dominant familial short stature (also known as dominant hereditary short stature), or idiopathic short stature. In various embodiments, short stature or growth plate disorders are the result of mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin-like growth factor 1 receptor (IGF1R).

[0073] In various embodiments, CNP variants can be used to treat growth plate disorders and short stature, including familial short stature, dominant familial short stature (also known as dominant hereditary short stature), or idiopathic short stature. In various embodiments, short stature or growth plate disorders are the result of mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, SHOX, NPR2, NPPC, FGFR3, or insulin-like growth factor 1 receptor (IGF1R).

[0074] In various embodiments, growth plate syndrome or short stature is associated with one or more mutations in RAS protein disease-related genes.

[0075] In various embodiments, CNP variants can be used to treat subjects with short stature whose height SDS is less than -1.0, -1.5, -2.0, -2.5, or -3.0, and at least one of their parents has a height SDS less than -1.0, -1.5, -2.0, or -2.5, optionally wherein the other parent's height is within the normal range. In various embodiments, CNP variants can be used to treat subjects with short stature whose height SDS is less than -1.0, -1.5, -2.0, -2.25, -2.5, or -3.0, and at least one of their parents has a height SDS less than -1.0, -1.5, -2.0, -2.25, or -2.5, optionally wherein the other parent's height is within the normal range. In various embodiments, CNP variants can be used to treat subjects with short stature whose height SDS is between -2.0 and -3.0. In various embodiments, CNP variants can be used to treat subjects with short stature whose height SDS is between -2.0 and -2.5. In various embodiments, short stature is associated with mutations in one or more genes related to short stature, such as collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin-like growth factor 1 receptor (IGF1R), or combinations thereof. In various embodiments, CNP variants can be used to treat subjects with short stature whose height SDS is between -2.0 and -2.5. In various embodiments, short stature is associated with mutations in one or more genes related to short stature, such as collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, SHOX, NPR2, NPPC, FGFR3, or insulin-like growth factor 1 receptor (IGF1R), or combinations thereof. In various embodiments, growth plate disorders or short stature are associated with mutations in one or more genes related to RAS protein disorders.

[0076] In various embodiments, short stature was determined by a polygenic risk score (PRS) to be the result of mutations in multiple genes. In various embodiments, the subject had an NPR2 mutation and low PRS. In various embodiments, the subject had an FGFR3 mutation and low PRS. In various embodiments, the subject had an NPR2 mutation and low PRS. In various embodiments, the subject had an IGF1R mutation and low PRS. In various embodiments, the subject had an NPPC mutation and low PRS. In various embodiments, the subject had a SHOX mutation and low PRS. In various embodiments, the subject had one or more mutations in one or more of FGFR3, IGF1R, NPPC, NPR2, and SHOX, and low PRS. In various embodiments, the PRS score was 1 or 2. In various embodiments, the PRS score was 1. In various embodiments, the PRS score was 2. The polygenic risk score (PRS) was calculated for height as described in Example 4. PRS 1 refers to the shortest height, and PRS 5 refers to the tallest height.

[0077] Also provided is a method for elongating bones or increasing long bone growth in a subject in need, the method comprising administering the composition described herein to the subject, wherein the administration elongates bones or increases long bone growth.

[0078] In various embodiments, the composition is administered subcutaneously, intradermally, intra-articularly, orally, or intramuscularly.

[0079] In various embodiments, the composition provides a prolonged release composition.

[0080] In various embodiments, the composition is applied once every 5 days, once a week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months.

[0081] In various embodiments, the administration increases the subject's annualized growth rate (AGV) at 12 months, optionally compared to baseline or a normal control. In various embodiments, the subject's AGV increases over one year or two years or longer.

[0082] In various embodiments, the administration resulted in an improvement in height Z-score at 12 months, optionally compared to baseline or a normal control.

[0083] In various embodiments, the subject is older than 3 years old. In various embodiments, the subject is between 3 and 17 years old. In various embodiments, the subject has an open epiphysis.

[0084] In various embodiments, the composition is administered at a dose of about 5 μg / kg to 500 μg / kg or about 15 μg / kg to 350 μg / kg. In various embodiments, the CNP prodrug is administered at a dose of about 5 μg / kg to 500 μg / kg, about 15 μg / kg to 350 μg / kg, about 25 μg / kg to 300 μg / kg, about 50 μg / kg to 250 μg / kg or about 75 μg / kg to 200 μg / kg. In various embodiments, the CNP variant is present at about 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 35 μg / kg, 40 μg / kg, 45 μg / kg, 50 μg / kg, 60 μg / kg, 70 μg / kg, 75 μg / kg, 80 μg / kg, 90 μg / kg, 100 μg / kg, 125 administered at doses of μg / kg, 150 μg / kg, 175 μg / kg, 200 μg / kg, 225 μg / kg, 250 μg / kg, 275 μg / kg, 300 μg / kg, 325 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, or 500 μg / kg.

[0085] In various embodiments, the administration does not cause cardiovascular (CV) side effects. In various embodiments, the CV side effects are changes in systemic blood pressure, mean arterial pressure, systolic and / or diastolic blood pressure, pulse pressure, or heart rate. In various embodiments, subjects receiving the CNP prodrug described herein to treat skeletal dysplasia have reduced or no cardiovascular (CV) side effects, such as changes in systemic blood pressure (mean arterial pressure, systolic and diastolic blood pressure, pulse pressure), and heart rate as observed with non-prodrug CNP variants.

[0086] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37) (SEQ ID NO: 1).

[0087] In various embodiments, the peptide further comprises an acetyl group. In various embodiments, the acetyl group is located at the N-terminus of the peptide. In various embodiments, the peptide further comprises an OH or NH2 group at the C-terminus. In various embodiments, the variants comprise one or more linker groups described herein. In various embodiments, the linker is a hydrolyzable linker.

[0088] In various embodiments, this disclosure provides a method for elongating or increasing long bone growth in a subject in need, the method comprising administering to the subject a composition comprising a CNP variant described herein, and wherein the administration elongates or increases long bone growth.

[0089] In various embodiments, the composition is administered subcutaneously, intradermally, intra-articularly, orally, or intramuscularly.

[0090] In various embodiments, the composition is applied once daily, once weekly, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months.

[0091] In various embodiments, the composition is a prolonged release composition.

[0092] Additionally, a method for treating a CNP-responsive condition or symptom is included, the method comprising administering to a subject a CNP variant or composition described herein, and monitoring the level of at least one bone-related or cartilage-related biomarker in the subject, wherein an increase in the level of the at least one bone-related or cartilage-related biomarker would indicate a therapeutic effect of the CNP peptide or variant on the subject or condition or symptom.

[0093] It also covers a method for overcoming cell growth arrest induced by constitutively active mutant fibroblast growth factor receptor 3 (FGFR-3), the method comprising contacting cells expressing constitutively active FGFR-3 with the CNP variant or composition described herein.

[0094] It also covers a method for stimulating cGMP production in cells expressing natriuretic natriuretic peptide receptor B (NPR-B), the method comprising contacting NPR-B-expressing cells with a CNP variant or composition described herein.

[0095] In various embodiments, the method further includes adjusting the dosage or frequency of administration of the CNP peptide or variant described herein, wherein i) the dosage or frequency of administration of the CNP peptide or variant is increased if the level of at least one bone-related or cartilage-related biomarker is below a target level; or ii) the dosage or frequency of administration of the CNP peptide or variant is decreased if the level of at least one bone-related or cartilage-related biomarker is above a target level.

[0096] In various embodiments, the at least one bone-related or cartilage-related biomarker is selected from the group consisting of: CNP, cGMP, type II collagen propeptide and fragment thereof, type II collagen and fragment thereof, type I collagen C-terminal peptide (CTx), osteocalcitonin, proliferating cell nuclear antigen (PCNA), type I collagen precursor propeptide (PINP) and fragment thereof, type I collagen and fragment thereof, chondroitin sulfate, collagen X, and alkaline phosphatase.

[0097] In various embodiments, the at least one bone-related or cartilage-related biomarker is selected from the group consisting of: CNP, cGMP, type II collagen propeptide and fragment thereof, type II collagen and fragment thereof, type I collagen C-terminal peptide (CTx), osteocalcitonin, proliferating cell nuclear antigen (PCNA), type I collagen propeptide and fragment thereof, type I collagen and fragment thereof, agglutinin sulfate chondroitin, collagen X, CXM (non-collagen 1 (NC1) domain of type X collagen), NTproCNP, alkaline phosphatase, N-terminal type I collagen propeptide, bone-specific alkaline phosphatase, type I collagen N-terminal propeptide / type I collagen propeptide (PINP), cross-linked type I collagen N-terminal peptide (NTx) tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomics readouts (e.g. from PAXgene® RNA), and CNP-variant bioactivity. Cartilage-related and bone-related biomarkers can be measured in any suitable biological sample, including but not limited to tissue, blood, serum, plasma, cerebrospinal fluid, synovial fluid, and urine. In some embodiments, the biomarkers are measured in blood, plasma, or serum from subjects undergoing in vivo efficacy / pharmacodynamic studies and / or from conditioned media from in vitro studies.

[0098] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37) (SEQ ID NO: 1).

[0099] In various embodiments, the peptide further comprises an acetyl group. In various embodiments, the acetyl group is located at the N-terminus of the peptide. In various embodiments, the acetyl group is located on an amino acid side chain within the peptide sequence. In various embodiments, the peptide further comprises an OH or NH2 group at the C-terminus. In various embodiments, the variant comprises one or more linker groups described herein. In various embodiments, the linker is a hydrolyzable linker.

[0100] A method for preparing the CNP variants described herein is also provided, the method comprising synthesizing peptides on a solid-phase resin using Fmoc amino acids.

[0101] In various embodiments, the method includes acetylation of the peptide by reacting a resin with NMP / Ac2O / DIEA (10:1:0.1, v / v / v).

[0102] In various embodiments, the method includes conjugating the peptide with a conjugation moiety optionally present on a lysine residue. In various embodiments, the method includes cleaving a protective amino group on the lysine residue, reacting the peptide with 2× Fmoc-aminoPEG(2), followed by a reaction with an amino acid, and then conjugating a lipid or fatty acid moiety.

[0103] It should be understood that each feature, embodiment, or combination described herein is a non-limiting illustrative example of any aspect of the invention and therefore means that it can be combined with any other feature, embodiment, or combination described herein. For example, when a feature is described using language such as “one embodiment,” “some embodiments,” “certain embodiments,” “other embodiments,” “specific exemplary embodiments,” and / or “another embodiment,” each of these types of embodiments is a non-limiting example of the feature intended to be combined with any other feature or combination of features described herein, without necessarily listing every possible combination.

[0104] These features or combinations of features are applicable to any aspect of the invention. In the case of instances of values ​​falling within the range disclosed, any one of these instances is covered as a possible endpoint of the range, covering any and all numerical values ​​between these endpoints, and any and all combinations of the upper limit endpoint and the upper limit endpoint are contemplated. Attached Figure Description

[0105] Figure 1 This illustrates the use of peptide-like or electronic linkers in the CNP conjugates described herein.

[0106] Figure 2 shows the stability of the CNP variant in human plasma for 24 hours.

[0107] Figure 3 The stability of the CNP variant under different culture conditions is demonstrated.

[0108] Figure 43 shows the effect of CNP variants (Pro-Gly-CNP) measured by cGMP stimulation on cells carrying NPR2 homozygous or heterozygous mutations.

[0109] Figure 5 The nucleotide and predicted protein sequence of the first exon in an NPR2 mutant clone transfected into RCS cells are shown.

[0110] Figure 6 This study presents exemplary NPR2 mutations related to the analyzed response to CNP.

[0111] Figure 7 Exemplary mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX are shown.

[0112] Figures 8A-8F The combined effect of PRS and rare coding variants on height is shown. Figure 8A The effect on height was assessed as a quantitative trait, and samples were divided into five groups based on their PRS (Personal Sequence Ratio), as shown in the violin plot, where the horizontal lines represent the 25th, 50th, and 75th percentiles of height. Samples were grouped according to missense, loss-of-function, or carrier status of any of the five core genes. Figure 8B The effect is reflected by the odds ratio for "idiopathic short stature" or ISS. PRS=3 is used as a reference for the ISS ratio relative to other PRS groups. Figure 8C Use PRS=1 noncarriers as a reference relative to the ISS ratio of missense and / or loss-of-function variants in the core gene. Figure 8D Use PRS=2 noncarriers as a reference relative to the ISS ratio of missense and / or loss-of-function variants in the core gene. Figure 8E Use PRS=3 noncarriers as a reference relative to the ISS ratio of missense and / or loss-of-function variants in the core gene. Figure 8F Use PRS=4 noncarriers as a reference relative to the ISS ratio of missense and / or loss-of-function variants in the core gene.

[0113] Figure 9 The mechanism of connector rupture is illustrated in one embodiment of this disclosure.

[0114] Figure 10 The components of the CNP variant shown include CNP, lipids, and linkers.

[0115] Figure 11 The structure and chemical name of the CNP variants disclosed in this paper are shown.

[0116] Figure 12 A schematic diagram illustrating the Phase 1 dosing study of CNP prodrug in healthy volunteers.

[0117] Figure 13 Visual appearance of the citrate-based formulation of the CNP prodrug.

[0118] Figure 14The results show the content of CNP variants over time when stored in different formulation buffers within a temperature range of -20 to 37°C.

[0119] Figure 15 This displays the amount of free drug released from different formulation buffers over time within a specific temperature range.

[0120] Figure 16 This demonstrates the junction area of ​​drug conjugates over time when stored in different formulation buffers within a certain temperature range.

[0121] Figure 17 The content of CNP conjugates was analyzed over time when stored in different formulation buffers within a certain temperature range.

[0122] Figure 18 This demonstrates the concentration of variants measured by UV over time when stored in different formulation buffers.

[0123] Figure 19 The variation of variant content over time in formulations using different excipients (histidine, glycine, and sorbitol) at pH 5.5 or 6 is shown.

[0124] Figure 20 The results of the feasibility study for manufacturing CNP prodrugs are presented.

[0125] Figure 21 Batch analysis of CNP prodrugs formulated with histidine or acetate buffers in different ratios (4:1 or 1:4, respectively) in trehalose and mannitol is presented.

[0126] Figure 22 The area under the curve after reversed-phase chromatography is shown.

[0127] Figure 23 The in-use stability and free drug content are demonstrated over time within a specific temperature range and pH 5.2 to 6.0.

[0128] Figure 24 Visual comparison of CNP prodrugs in different formulations.

[0129] Figure 25 shows the results of WT mice and Raf1 mice after 6 weeks of treatment. + / L613V Nose-to-anus length in mice.

[0130] Figure 26This section shows the nose-to-anus length of WT and Rit+ / - mice treated for 10 weeks. IP, intraperitoneal; MEKi, mitogen-activated protein kinase inhibitor; NA, nose-anus; QAD, every other day; QD, once daily; SC, subcutaneous; Veh., mediator; WT, wild-type. # Significant differences from the corresponding mediator treatment group were determined by unpaired t-test. * Significant differences from the genotype-matched group treated with CNP were determined by unpaired t-test.

[0131] Figure 27 Whole-body μCT images of WT mice treated with a cauterizer (left image) or a CNP prodrug (right image). μCT stands for Microcomputed Tomography; WT stands for Wild-type.

[0132] Figure 28 The plasma pharmacokinetics of the CNP prodrug and released vosoritide in mice following a single SC administration are demonstrated.

[0133] Figure 29 The mean (±SD) plasma vosolidide and CNP prodrug concentration-time curves are shown after a single dose of CNP prodrug administration.

[0134] Figure 30 A and 30B show the levels of CNP prodrug in the plasma of NHP after administration of CNP prodrug at IV or SC. Figure 30 A) and vosolamide ( Figure 30 The curve showing the change of the average (±SD) concentration of B over time.

[0135] Figure 31 A and 31B show the levels of CNP prodrug in the plasma of male and female NHPs after administration of CNP prodrug at IV or SC. Figure 31 A) and vosolamide ( Figure 31 The curve showing the change of the average (±SD) concentration of B over time.

[0136] Figure 32A The mean (±SD) plasma vosolidin and CNP prodrug concentration-time curves and dose-normalized mean plasma vosolidin and CNP prodrug concentration-time curves are shown. Figure 32B The mean (±SD) plasma vosolidin and CNP prodrug concentration-time curves and dose-normalized mean plasma vosolidin and CNP prodrug concentration-time curves are shown.

[0137] Figure 33 The mean (±SD) plasma vosolidide and CNP prodrug concentration-time curves are shown for day 1 and day 22.

[0138] Figure 34The demonstration shows the binding of CNP prodrugs with NPR-B and NPR-C compared to BMN111. Detailed Implementation

[0139] This disclosure relates to stable CNP variants that can be used to treat skeletal dysplasia and bone growth disorders.

[0140] Unless the context clearly specifies otherwise, as used in this specification and the appended claims, the indefinite articles “a” and “an” and the definite article “the” include both plural and singular indicators.

[0141] The term "about" or "approximately" refers to an acceptable error in the measurement of a particular value as determined by a person skilled in the art, depending in part on how the value is measured or determined. In some embodiments, the term "about" or "approximately" refers to a range of 1, 2, 3, or 4 standard deviations. In some embodiments, the term "about" or "approximately" refers to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. When the term "about" or "approximately" precedes the first value in a series of two or more values, it should be understood that the term "about" or "approximately" applies to each value in the series.

[0142] The term "C-type natriuretic peptide" or "CNP" refers to a small, single-chain peptide with a 17-amino acid ring structure at its C-terminus (for CNP precursor proteins, i.e., NPPC, GenBank accession number NP_077720) and its variants. The 17-mer CNP ring structure is also referred to as CNP 17, the CNP ring, or the CNP ring domain. CNPs comprise an active peptide containing 53 amino acids (CNP-53) and a mature peptide containing 22 amino acids (CNP-22), as well as peptides of varying lengths between these two peptides.

[0143] In various embodiments, the “CNP variant” shares at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% homology with the wild-type NPPC within the same number of amino acid residues. Further consideration is given that the CNP variant peptide may contain about 1 to about 53, or 1 to 39, or 1 to 38, or 1 to 37, or 1 to 35, or 1 to 34, or 1 to 31, or 1 to 27, or 1 to 22, or 10 to 35, or about 15 to about 37 residues of the NPPC polypeptide. In one embodiment, the CNP variant may comprise a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 amino acids derived from the NPPC polypeptide.

[0144] The term "conjugated moiety" refers to the portion conjugated to the variant peptide. Conjugated moieties include lipids, fatty acids, hydrophilic spacers, synthetic polymers, linkers, or optionally combinations thereof.

[0145] The term "effective dose" refers to a dose sufficient to produce the desired outcome for a subject's health condition, lesion, or disease, or sufficient for diagnostic purposes. The desired outcome may include subjective or objective improvement in the dose recipient. "Therapeutic effective dose" refers to a dose of medicine that effectively produces the expected beneficial effect on health. The appropriate "effective" dose in any individual case can be determined by a person skilled in the art using routine laboratory methods. It should be understood that the specific dose level and frequency for any particular patient can vary and will depend on a variety of factors, including the activity of the specific compound used; the bioavailability, metabolic stability, excretion rate, and duration of action of said compound; the administration pattern and timing of the compound; the patient's age, weight, general health condition, sex, and diet; and the severity of the specific condition.

[0146] "Largely pure" or "isolated" means that the target species is the dominant species present (i.e., more abundant, by molar number, than any other individual macromolecular species in the composition), and that the substantially purified portion is a composition in which the target species constitutes at least about 50% (by molar number) of all macromolecular species present. In one embodiment, a substantially pure composition means that the species of interest constitutes at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or a higher percentage, by molar number or weight, of the macromolecular species present in the composition. If the composition consists substantially of a single macromolecular species, the target species is purified to be substantially homogeneous (contaminant species are undetectable in the composition by conventional detection methods). For the purposes of this definition, solvent species, small molecules (<500 Daltons), stabilizers (e.g., BSA), and elemental ionic species are not considered macromolecular species. In one embodiment, the compounds of this disclosure are substantially pure or isolated. In another embodiment, the compounds of this disclosure are substantially pure or isolated relative to the macromolecular starting materials used in their production. In yet another embodiment, the pharmaceutical composition of this disclosure comprises a substantially pure or isolated CNP variant mixed with one or more pharmaceutically acceptable excipients, carriers or diluents and optionally with another bioactive agent.

[0147] "Treatment" refers to preventive treatment, therapeutic treatment, or diagnostic treatment. In some embodiments, "treatment" means administering a compound or composition to a subject for therapeutic, preventive, or diagnostic purposes.

[0148] "Preventative" treatment is a treatment administered to a subject who does not show signs of disease or only shows early signs of disease, with the aim of reducing the risk of developing a lesion. The compounds or compositions disclosed herein may be provided as preventative treatment to reduce the likelihood of developing a lesion or to minimize the severity of a lesion, if it has already developed.

[0149] "Therapeutic" treatment is treatment administered to a subject exhibiting signs or symptoms of a lesion for the purpose of alleviating or eliminating such signs or symptoms. These signs or symptoms can be biochemical, cellular, histological, functional or physical, subjective or objective. The compounds disclosed herein may also be provided as a therapeutic treatment or for diagnostic purposes.

[0150] "Diagnosis" refers to the identification of the presence, extent, and / or nature of a pathological condition. Diagnostic methods vary in specificity and selectivity. While a particular diagnostic method may not provide a definitive diagnosis of a certain condition, it is sufficient if the method provides positive indications that aid in diagnosis.

[0151] "Bone-related or cartilage-related biomarkers" or "bone-related or cartilage-related markers" refer to growth factors, enzymes, proteins, or other detectable biological substances or components whose levels increase or decrease with, for example, cartilage turnover, cartilage formation, cartilage growth, bone resorption, bone formation, bone growth, or combinations thereof. Such biomarkers can be measured before, during, and / or after administration of the CNP variants described herein. Exemplary bone-related or cartilage-related biomarkers include, but are not limited to, CNP, cGMP, type II collagen propeptide and fragments, type II collagen and fragments, type I collagen propeptide and fragments, type I collagen and fragments, osteocalcitonin, proliferating cell nuclear antigen (PCNA), chondroitin sulfate, collagen X, basic propeptide and fragments of type I collagen, type I collagen and fragments, chondroitin sulfate, collagen X, CXM (non-collagen 1 (NC1) domain of type X collagen), NTproCNP, N-terminal type I collagen propeptide, bone-specific alkaline phosphatase, N-terminal propeptide of type I collagen / type I collagen N-propeptide (PINP), cross-linked type I collagen C-terminal peptide (CTx), cross-linked type I collagen N-terminal peptide (NTx), tartrate-resistant acid phosphatase 5b (TRAP-5b), and transcriptomic readouts (e.g., from PAXgene®). RNA) and CNP-variant bioactivity. Cartilage-related and bone-related biomarkers can be measured in any suitable biological sample, including but not limited to tissue, blood, serum, plasma, cerebrospinal fluid, synovial fluid, and urine. In some embodiments, biomarkers are measured in blood, plasma, urine, or serum from subjects undergoing in vivo efficacy / pharmacodynamic studies and / or from conditioned media from in vitro studies.

[0152] "Pharmaceutical composition" or "formulation" means a composition suitable for medicinal use in test animals, including humans and mammals. A pharmaceutical composition comprises a therapeutically effective amount of a CNP variant, optionally another bioactive agent, and optionally a pharmaceutically acceptable excipient, carrier, or diluent. In one embodiment, a pharmaceutical composition encompasses compositions comprising one or more active ingredients and one or more inert ingredients constituting a carrier, as well as any product directly or indirectly produced by the combination, compounding, or aggregation of any two or more ingredients, or by the dissociation of one or more ingredients, or by other types of reactions or interactions of one or more ingredients. Therefore, pharmaceutical compositions of this disclosure encompass any composition prepared by mixing the compounds of this disclosure with a pharmaceutically acceptable excipient, carrier, or diluent.

[0153] "Pharmaceutically acceptable carrier" refers to any of the standard drug carriers, buffers, etc., such as phosphate-buffered saline solutions, 5% dextran solutions, and emulsions (e.g., oil / water or water / oil emulsions). Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifiers, wetting agents, lubricants, flow aids, sweeteners, flavoring agents, and coloring agents. Suitable drug carriers, excipients, and diluents are described in Remington's Pharmaceutical Sciences, 19th edition (Mack Publishing Co., Easton, 1995). Preferred drug carriers depend on the intended administration mode of the active agent. Typical administration modes include enteral (e.g., oral) or parenteral administration (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection; or local, percutaneous, or mucosal administration).

[0154] "Pharmaceutical acceptable salts" are salts that can be formulated into compounds for medicinal use, including but not limited to metal salts (such as sodium, potassium, magnesium, calcium salts, etc.) and salts of ammonia or organic amines.

[0155] "Pharmaceutical acceptable" or "pharmacologically acceptable" means a substance that is not biologically or otherwise undesirable, that is, a substance that can be administered to an individual without causing any undesirable biological effects or interacting in a harmful manner with any component of a composition containing it or with any component present on or in the body of an individual.

[0156] "Physiological conditions" refers to the conditions of an animal's (e.g., human) body. Physiological conditions include, but are not limited to, body temperature and an aqueous environment with physiological ionic strength, pH, and enzymes. Physiological conditions also encompass conditions within a specific subject that differ from the "normal" conditions present in most subjects, such as a temperature different from the normal human body temperature of approximately 37°C or a blood pH different from the normal human blood pH of approximately 7.4.

[0157] "Physiological pH" or "pH within the physiological range" refers to a pH in the range of approximately 7.0 to 8.0 (inclusive), and more commonly in the range of approximately 7.2 to 7.6 (inclusive).

[0158] As used herein, the term "subject" encompasses both mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammal class: humans; non-human primates such as chimpanzees, and other ape and monkey species; livestock such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc. The term does not indicate a specific age or sex. In various embodiments, the subject is a human. In various embodiments, the subject is a child or adolescent. In various embodiments, the subject is an infant. In various embodiments, the subject is older than 3 months, older than 2 months, older than 1 month, or older than 6 months.

[0159] C-type natriuretic peptide variant

[0160] C-type natriuretic peptide (CNP) (Biochemistry and Biophysics Research Communications, 168: 863-870 (1990) (For the CNP precursor protein, NPPC, GenBank accession number NP_077720) (Journal of Hypertension, 10: 907-912 (1992)) is a small single-chain peptide belonging to the peptide family (ANP, BNP, CNP) with a 17-amino acid ring structure (Levin et al., The New England Journal of Medicine, 339: 863-870 (1998)) and plays an important role in a variety of biological processes. CNP interacts with natriuretic peptide receptor-B (NPR-B, GC-B) to stimulate the production of cyclic guanosine monophosphate (cGMP) (Journal of Hypertension, 10: 1111-1114 (1992)). CNP is widely expressed, including in the central nervous system, reproductive tract, bone, and vascular endothelium (Hypertension, 10: 907-912 (1992)). 49: 419-426 (2007)).

[0161] Natural CNP genes and peptides have been previously described. U.S. Patent No. 5,352,770 discloses CNP-22, a sequence identical to human CNP, isolated and purified from pig brain, and its use in the treatment of cardiovascular indications. U.S. Patent No. 6,034,231 discloses the human gene and peptide of the proto-CNP (126 amino acids) and the human CNP-53 gene and peptide. Mature CNP is a peptide of 22 amino acids (CNP-22). Certain CNP variants are disclosed in U.S. Patent No. 8,198,242, which is incorporated herein by reference.

[0162] In various embodiments, the CNPs of this disclosure include truncated CNPs ranging from human CNP-17 (hCNP-17) to human CNP-53 (hCNP-53) and having a wild-type amino acid sequence derived from hCNP-53. Such truncated CNP peptides include:

[0163] DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-53) (SEQ IDNO: 56); LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-52) (SEQ IDNO: 15); RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-51) (SEQ IDNO: 16);

[0164] VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-50) (SEQ IDNO:17);

[0165] DTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-49) (SEQ ID NO: 18);

[0166] TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-48) (SEQ ID NO:19);

[0167] KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-47) (SEQ ID NO:20);

[0168] SRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-46) (SEQ ID NO:21);

[0169] RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-45) (SEQ ID NO:22);

[0170] AAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-44)(SEQ ID NO:23);AWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-43)(SEQ ID NO:24);WARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-42)(SEQ ID NO:25);ARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-41)(SEQ ID NO:26);RLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-40)(SEQ ID NO:27);LLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-39)(SEQ ID NO:28);LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38)(SEQ IDNO:2);QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-37)(SEQ ID NO:3);EHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-36)(SEQ ID NO:29);HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-35)(SEQ ID NO:30);PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-34)(SEQ ID NO:4);NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-33)(SEQ ID NO:31);ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-32)(SEQ ID NO:32);

[0171] RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-31) (SEQ ID NO:33); KYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-30) (SEQ ID NO:34); YKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-29) (SEQ ID NO:35); KGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-28) (SEQ ID NO:36); GANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-27) (SEQ ID NO:37); ANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-26) (SEQ ID NO:38); NKKGLSKGCFGLKLDRIGSMSGLGC (CNP-25) (SEQ ID NO:39);KKGLSKGCFGLKLDRIGSMSGLGC (CNP-24) (SEQ ID NO:40); KGLSKGCFGLKLDRIGSMSGLGC (CNP-23) (SEQ ID NO:41); GLSKGCFGLKLDRIGSMSGLGC (CNP-22) (SEQ ID NO: 68); LSKGCFGLKLDRIGSMSGLGC (CNP-21) (SEQ ID NO:42); SKGCFGLKLDRIGSMSGLGC (CNP-20) (SEQ ID NO: 43); KGCFGLKLDRIGSMSGLGC (CNP-19) (SEQ ID NO: 44); GCFGLKLDRIGSMSGLGC (CNP-18) (SEQ ID NO: 45); and CFGLKLDRIGSMSGLGC (CNP-17) (SEQ ID NO: 67).

[0172] In various embodiments, the CNP variant peptide is a modified CNP-37 or CNP-38 peptide that optionally has one or more mutations / substitutions at the furin cleavage site (underlined) and / or contains glycine or proline-glycine at the N-terminus. Exemplary CNP-37 variants include, but are not limited to:

[0173] QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID NO: 46]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID NO: 47); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID NO: 48); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N); SEQ ID NO: 49];

[0174] PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID NO:1); MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID NO: 50);

[0175] GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO:51) GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:52); GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:53); GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (SEQ IDNO:54); and GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:55).

[0176] In various embodiments, the CNP variants of the present disclosure include PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5);

[0177] PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6);

[0178] PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5);

[0179] PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7);

[0180] PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-YGIU-C 18DA)LDRIGSMSGLGC(SEQID NO:5); and

[0181] PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-YGIU-C18DA)LDRIGSMSGLGC(SEQ IDNO: 1).

[0182] In various embodiments, the CNP variant also includes an acetyl group. In various embodiments, the acetyl group is located at the N-terminus, the C-terminus, or attached to an internal amino acid side group. In various embodiments, the acetyl group is located at the N-terminus of the peptide.

[0183] In various embodiments, the peptide variants further include an OH or NH2 group at the C-terminus.

[0184] In various embodiments, the CNP variants are selected from the group consisting of:

[0185] Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 8),

[0186] Ac-PGQEH PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 9),

[0187] Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10),

[0188] AC-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 11), and

[0189] AC-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 12).

[0190] In various embodiments, the CNP variants are selected from the group consisting of: PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 8), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 9), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 11), PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 12), and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 7).

[0191] In various embodiments, the CNP variants are selected from the group consisting of:

[0192] Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 8);

[0193] Ac-PGQEH PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 9);

[0194] Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10);

[0195] Ac-PGQEH PNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 11);

[0196] Ac-PGQEH PQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 12);

[0197] Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 13); and

[0198] Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 14).

[0199] In various embodiments, the CNP variant is selected from the group consisting of: Ac-PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-YGIU-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 8). In various embodiments, the CNP variant is Ac-PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-YGIU-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 1). In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-YGIU-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 1).

[0200] In another embodiment, for any of the CNP variants described herein having one or more asparagine (Asn / N) residues and / or one or more glutamine (Gln / Q) residues, regardless of whether they have a wild-type sequence or a non-natural amino acid sequence, any one or more Asn residues and / or any one or more Gln residues may be independently substituted with any other natural or non-natural amino acid, including conserved substitutions, such as Asn to Gin. One or more such substitutions are partly designed to minimize or avoid any potential deamidation of asparagine and / or glutamine.

[0201] In one embodiment, the CNP variant is transmitted via Cys 6 With Cys 22 Cys form disulfide bonds and cyclize, as indicated in the wtCNP22 peptide. 6 It can be a cysteine ​​analog, such as homocysteine ​​or penicillamine. In another embodiment, the CNP variant can be cyclized by forming covalent bonds head-to-tail, side-to-side, side-to-head, or side-to-tail. In one embodiment, the covalent bond is formed between an N-terminal or N-terminal amino acid and a C-terminal or C-terminal amino acid (in this case referred to as the "terminal" amino acid). In another embodiment, the covalent bond is formed between the side chains of two terminal amino acids. In yet another embodiment, the covalent bond is formed between the side chain of one terminal amino acid and the end group of another terminal amino acid, or between the end groups of two terminal amino acids.

[0202] Head-to-tail cyclization of the terminal amine and terminal carboxyl group can be carried out using a variety of methods, such as using p-nitrophenyl ester, 2,4,5-trichlorophenyl ester, pentafluorophenyl ester, azid method, mixed anhydride method, HATU, carbodiimide (e.g., DIC, EDC or DCC) in the presence of catalysts such as HOBt, HONSU or HOAt or on resin.

[0203] Additionally, cyclic structures can be formed via bridging groups on the side chains of amino acid residues and / or terminal amino acid residues involving CNP variants. A bridging group is a chemical part that cyclizes two parts of the peptide. Non-limiting examples of bridging groups include amides, thioethers, thioesters, disulfides, ureas, carbamates, sulfonamides, etc. Various methods for incorporating units having such bridging groups are known in the art. For example, a lactam bridge (i.e., a cyclic amide) can be formed between an N-terminal amino group or an amino group on a side chain and a C-terminal carboxylic acid group or a carboxyl group on a side chain, for example, between the side chain of lysine or ornithine and the side chain of glutamic acid or aspartic acid. Thioesters can be formed between a C-terminal carboxyl group or a carboxyl group on a side chain and a thiol group on the side chain of cysteine ​​or a cysteine ​​analog.

[0204] Alternatively, crosslinking can be achieved by incorporating lanethionine (thiodialanine) residues to link alanine residues together via thioether bonds. In another approach, a crosslinking agent, such as a dicarboxylic acid (e.g., succinic acid (octanoic acid)), can link functional groups, such as free amino, hydroxyl, and thiol groups, to the amino acid side chains.

[0205] Enzymatic cyclization can also be used. For example, the thioesterase domain of tyrocidine synthase has been reported to be used for the cyclization of thioester precursors, subtilisin mutants for the cyclization of glycolate phenylalanylamide ester, and antibody ligase 16G3 for the cyclization of p-nitrophenyl ester. For a review of peptide cyclization, see Davies, *Journal of Peptide Sci.*, 9: 471-501 (2003), which is incorporated herein by reference in its entirety.

[0206] In some embodiments, the final product has a purity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least about 99%.

[0207] peptide conjugates

[0208] Peptide therapeutics are attractive biotherapeutic agents, but are often hampered by low stability in solution and short half-lives (Tang et al., *Eur J Pharm Sci.* 102:63-70, 2017). Attempts to improve the efficacy of peptide therapeutics by enhancing stability and / or prolonging half-life include encapsulating hydrophilic peptides into biodegradable particles such as liposomes or polymer particles. However, this attempt is difficult to achieve due to the cationic nature of these peptides and their ability to electrostatically interact with negatively charged polymer liposomes (Griesser et al., *Int J Pharmaceutics* 520:267-274, 2017). The generation of peptide conjugates has emerged as a means to better encapsulate hydrophilic polymers in microparticles or liposomes (Lu et al., *Molecular Pharmaceutics* 15:216-225, 2018).

[0209] A peptide can be an amino acid chain having 5 to 100 amino acids. A peptide can contain positively charged amino acids, negatively charged amino acids, or a mixture of both, such that the peptide can interact with charged moieties, such as cations, anions, or combinations thereof, which have a species with a charge opposite to that of the charged species in the peptide.

[0210] Consideration is given to complexing the peptide with a portion (e.g., a conjugated portion) that imparts increased stability or half-life. In various embodiments, the conjugated portion is complexed via non-covalent bonding or linked via covalent bonding. The portion may be non-covalently linked to the peptide via electrostatic interactions. Alternatively, the portion may be covalently linked to the peptide via one or more linker portions. Linkers can be cleavable or non-cleavable. Cleavable linkers can be cleaved by enzymes, nucleophiles / basics, reducing agents, photoirradiation, electrophiles / acids, organometallic and metallic reagents, or oxidizing agents. Linkers can also be self-degrading linkers. Linkers can also be traceless linkers. Exemplary linkers include, but are not limited to, N-succinimide-3-(2-pyridyldithiol)propionate (SPDP), iminothiacyclopentane (IT), bifunctional derivatives of imide esters (e.g., dimethyl diimide adipate hydrochloride), active esters (e.g., bissuccinimide octanoate), aldehydes (e.g., glutaraldehyde), diazidide compounds (e.g., bis(p-azidobenzoyl)hexamethylenediamine), diazido derivatives (e.g., bis(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and difluorinated compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene), β-alanine, 4-aminobutyric acid (GABA), 2-aminoethoxyic acid (AEA), aminoethoxy-2-ethoxyacetic acid (AEEA), 5-aminovaleric acid (AVA), 6-aminohexanoic acid (Abx), vicinal diol cleavable linkers, and trimethyl lactones. LockLactonization), p-alkoxyphenyl carbamate, bicin, peptide-like or bicin-type connectors, and the electronic connectors described herein.

[0211] In various embodiments, the linker connects to a residue of the CNP variant within the CNP cyclic domain or to a site other than the CNP cyclic domain. In various embodiments, the linker connects to a lysine residue. In various embodiments, the linker connects to a lysine residue within the CNP cyclic domain.

[0212] In various embodiments, the CNP variant is connected to the lacing portion via a connector. In various embodiments, the connector is connected to the lacing portion via a hydrophilic spacer.

[0213] In various embodiments, the connector is a hydrolyzable connector.

[0214] In various embodiments, the connector is a peptide-like connector or an electronic connector. In various embodiments, the connector is a peptide-like connector. In various embodiments, the connector is an electronic connector. In various embodiments, the connector includes an SO2 portion. An exemplary connector is shown. Figure 1 And in the following text. After further consideration, Figure 1The linker in the structure is modified by substitution on the R group. For example, the bicin-type linker includes the structure described below:

[0215]

[0216] In various embodiments, the portion conjugated to the peptide is a synthetic polymer, such as polyethylene glycol, a linker, a lipid moiety, or a fatty acid, or a combination thereof. In various embodiments, the CNP variant is conjugated to a fatty acid, an amino acid, a spacer, and a linker. In various embodiments, the CNP variant is conjugated to a fatty acid, an amino acid, a polyethylene glycol spacer, or a polyethylene glycol derivative spacer and linker. In various embodiments, the CNP variant is conjugated to a fatty acid, an amino acid, a spacer, and a linker, wherein the spacer comprises a substituted C-6 to C-20 alkyl chain or any amino acid, or a combination thereof, wherein the carbon atom of the alkyl chain may be replaced by one or more of O, NH, N (C-1 to C-6 alkyl), or a carbonyl group.

[0217] In various embodiments, the CNP variant is conjugated with a fatty acid. It is assumed that lipid technology increases the serum half-life of the CNP variant, allowing for lower frequency injections and / or improved oral delivery. In various embodiments, the fatty acid is a short-chain, medium-chain, long-chain fatty acid, or a dicarboxylic acid. In various embodiments, the fatty acid is a saturated or unsaturated fatty acid. In various embodiments, the fatty acid is a C-6 to C-20 fatty acid. In various embodiments, the fatty acid is a C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 fatty acid. In various embodiments, the fatty acid is decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, eicosanoic acid, or a diacid thereof. In various embodiments, the fatty acid is conjugated with a lysine residue.

[0218] In various embodiments, the CNP variants described herein are considered to include the conjugation moiety described herein. The conjugation moiety is considered to be located on a residue of a CNP cyclic domain or at a site other than a CNP cyclic domain. In various embodiments, the conjugation moiety is located on a lysine residue. In various embodiments, the conjugation moiety includes one or more acid moieties. In various embodiments, the acid moieties are fatty acids.

[0219] In various embodiments, the conjugated portion comprises an acidic portion linked to a hydrophilic spacer. In various embodiments, the hydrophilic spacer is a substituted C-6 to C-20 alkyl chain or any amino acid, or a combination of both, wherein the carbon atom of the alkyl chain may be replaced by one or more of O, NH, N (C-1 to C-6 alkyl), or a carbonyl group. In various embodiments, the hydrophilic spacer is any amino acid. In various embodiments, the hydrophilic spacer is γ-glutamic acid (yGlu). In various embodiments, the hydrophilic spacer is a substituted C-6 to C-20 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-9 to C-18 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-18 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-9 alkyl chain. In various embodiments, the hydrophilic spacer is one or more OEG (8-amino-3,6-dioxanoic acid) groups. In various embodiments, the hydrophilic spacer is one or two OEG (8-amino-3,6-dioxanoic acid) groups. In various embodiments, the hydrophilic spacer is OEG (8-amino-3,6-dioxanoic acid). In various embodiments, the spacer is OEG (8-amino-3,6-dioxanoic acid) or yGlu. In various embodiments, the hydrophilic spacer is γ-glutamic acid (yGlu) linked to one or more OEG (8-amino-3,6-dioxanoic acid) groups. In various embodiments, the hydrophilic spacer is γ-glutamic acid (yGlu) linked to one or two OEG (8-amino-3,6-dioxanoic acid) groups (diEG). In various embodiments, the acid moiety and the hydrophilic spacer have the structure AEEA-AEEA-yGlu-d8DA.

[0220] In various embodiments, CNP variants have the following structure:

[0221] PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-yGlu-C18DA)LDRIGSMSGLGC (SEQ ID NO: 5), or Ac-PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-yGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 8). In various embodiments, the CNP variant has the structure PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-yGlu-C18DA)LDRIGSMSGLGC, Ac-PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-yGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 1), or PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-yGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 1). In various embodiments, CNP variants include variants where the Asn of the aforementioned peptide is changed to Glu.

[0222] In various embodiments, this disclosure covers the use of hydrophilic or water-soluble polymers (e.g., alkyl oxide chains, where carbon atoms may be replaced by one or more oxygen atoms, such as polyethylene glycol (PEG) or polyethylene oxide (PEO), etc.). In various embodiments, the type of water-soluble polymer (e.g., homopolymer or copolymer; random, alternating, or block copolymer; linear or branched; monodisperse or polydisperse), bonds (e.g., hydrolyzable or stable bonds, such as amide, imine, acetal, alkylene, or ester bonds), conjugation sites (e.g., at the N-terminus, internal, and / or C-terminus), and length (e.g., from about 0.2, 0.4, or 0.6 kDa to about 2, 5, 10, 25, 50, or 100 kDa) can vary. As is known in the art, hydrophilic or water-soluble polymers can be conjugated to CNP variants by means of N-hydroxysuccinimide (NHS) or aldehyde-based chemicals or other chemicals. In various embodiments, negatively charged PEG-CNP variants may be designed to reduce renal clearance, including but not limited to the use of carboxylated, sulfated, and phosphorylated compounds (Caliceti, *Advanced Drug Delivery Review*, 55: 1261-77 (2003); Perlman, *Journal of Clinical Endocrinology and Metabolism*, 88: 3227-35 (2003); Pitkin, *Antimicrob. Ag. Chemo.*, 29: 440-444 (1986); Vehaskari, *Kidney International*, 22: 127-135 (1982)). In one embodiment, the PEG (or PEO) moiety contains one or more carboxyl groups, one or more sulfate groups, and / or one or more phosphate groups.

[0223] In another embodiment, the hydrophilic polymer (e.g., PEG or PEO) conjugated to the N-terminus, C-terminus, and / or one or more internal sites of the CNP variant described herein contains one or more functional groups that are positively charged under physiological conditions. Such portions are particularly designed to improve the distribution of such conjugated CNP variants in cartilage tissue. In one embodiment, the PEG portion contains one or more primary, secondary or tertiary amino, quaternary ammonium, and / or other amine-containing (e.g., urea) groups.

[0224] Preparation method

[0225] This document also covers a method for preparing a composition comprising a CNP variant and optionally comprising the conjugation portion described herein.

[0226] In various embodiments, the CNP variants were synthesized using standard protein synthesis chemistry methods. For example, peptides were synthesized stepwise using solid-phase resin and standard Fmoc chemistry methods. The peptides were cleaved from the resin using trifluoroacetic acid (TFA) and purified by reversed-phase high-performance liquid chromatography (RP-HPLC).

[0227] In various embodiments, the method further includes acetylation of the peptide by reacting the resin with NMP / Ac2O / DIEA optionally at a ratio of 10:1:0.1 (v / v / v).

[0228] Another method is provided in which the peptide is conjugated to a conjugation moiety optionally present on a lysine residue. The method includes cleaving a protective amino group on the lysine residue, reacting the peptide with 2× Fmoc-aminoPEG(2), followed by a reaction with an amino acid, and then conjugating a lipid or fatty acid moiety. In various embodiments, the conjugation moiety comprises one or more lipids or fatty acids, and a hydrophobic spacer.

[0229] The method also provides a step of cleaving the peptide from the resin by contacting it with trifluoroacetic acid, and a step of purifying the peptide by reversed-phase HPLC.

[0230] In some embodiments, the CNP variants described herein are generated via a recombination method comprising culturing host cells in a culture medium containing a first polynucleotide encoding a CNP variant polypeptide, optionally linked to a second polynucleotide encoding a cleavable peptide or protein, under conditions that enable the expression of a polynucleotide-encoded fusion polypeptide. In some embodiments, the host cells are transformed with an expression vector containing a polynucleotide encoding a CNP variant polypeptide, optionally linked to a polynucleotide encoding a cleavable peptide or protein. In some embodiments, the fusion polypeptide is expressed as a soluble protein or inclusion body. The expressed fusion polypeptide can be isolated from the host cells or culture medium, and the isolated fusion polypeptide can be contacted with a lysis agent to release the CNP variant.

[0231] U.S. Patent 8,198,242 discloses a method for preparing CNP variant peptides, including the use of host cells, expression vectors, cleavable peptides, and culture parameters, which is hereby incorporated by reference.

[0232] How to use

[0233] Achondroplasia results from an autosomal dominant mutation in the gene for fibroblast growth factor receptor 3 (FGFR-3), which causes abnormal cartilage formation. FGFR-3 normally has a negative regulatory effect on chondrocyte growth and therefore on bone growth. In achondroplasia, the mutant form of FGFR-3 is constitutively active, leading to severe bone shortening. In humans, activating mutations in FGFR-3 are a major cause of hereditary dwarfism. Mice with activated FGFR-3 serve as models of achondroplasia (the most common form of skeletal dysplasia), and overexpression of CNP rescues these animals from the effects of dwarfism. Therefore, functional variants of CNP are potential therapeutic agents for various skeletal dysplasias.

[0234] By stimulating chondrocyte matrix production, proliferation, and differentiation, and increasing long bone growth, the CNP variants disclosed herein can be used to treat mammals, including humans, suffering from bone-related disorders such as skeletal dysplasia. Non-limiting examples of CNP-responsive bone-related disorders and skeletal dysplasia include chondrodysplasia, decreased chondrogenesis, short stature, dwarfism, osteochondrodysplasia, lethal chondrodysplasia, and congenital osteoogenesis. Congenita, chondrodysplasia, congenital chondrodysplasia, homozygous chondrodysplasia, brachydactyly, congenital lethal hypophosphatase, perinatal lethal congenital osteogenic dysplasia, short rib polydactyly syndrome, limb root type congenital chondrodysplasia, Janssen type metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, osteodysplasia, malformation dysplasia, congenital short femur, Langer type midlimb dysplasia, Nivig type midlimb dysplasia, Robinnoc syndrome, Reinhart syndrome, acrodysplasia, peripheral osteodysplasia, Knifell's dysplasia, fibrocartilage hyperplasia, Roberts syndrome, acromegaly, microlimb syndrome, Moquer syndrome, Knifell's syndrome, variability dysplasia, and vertebral epiphyseal and metaphyseal dysplasia. The short stature, growth plate disorders, bone-related disorders, or skeletal dysplasia covered in this article include conditions associated with NPR2 mutations, SHOX mutations (Turner syndrome / Lreywell disease), and PTPN1 1 mutations (Nunnan syndrome).

[0235] By stimulating matrix production, proliferation, and differentiation of chondrocytes and increasing long bone growth, the CNP variants disclosed herein can be used to treat mammals, including humans, suffering from bone-related disorders such as skeletal dysplasia. Non-limiting examples of CNP-responsive bone-related disorders and skeletal dysplasia include achondroplasia, decreased chondrogenesis, short stature, dwarfism, osteochondrodysplasia, lethal chondrodysplasia, congenital osteogenic dysplasia, chondrodysplasia, congenital chondrodysplasia, homozygous chondrodysplasia, brachydactyly, congenital lethal hypophosphatase syndrome, perinatal lethal congenital osteogenic dysplasia, short rib polydactyly syndrome, limb root type congenital chondrodysplasia, and Janssen type metaphyseal dysplasia. This includes congenital vertebral epiphyseal dysplasia, osteodystrophy, malformation, congenital short femur, Langer type midlimb dysplasia, Nivig type midlimb dysplasia, Robinnoc syndrome, Reinhart syndrome, acrodysplasia, peripheral skeletal dysplasia, Knifell's dysplasia, fibrocartilage hyperplasia, Roberts syndrome, acromegaly, microlimb syndrome, Moquer syndrome, Knifell's syndrome, varicella dysplasia, and vertebral epiphyseal-metaphysema. The short stature, growth plate disorders, bone-related disorders, or skeletal dysplasia covered in this article include conditions associated with NPR2 mutations, SHOX mutations (Turner syndrome / Lerreville's disease), PTPN11 mutations (Nunnan syndrome), and IGF1R mutations.

[0236] This document further provides that the CNP variants disclosed herein can be used to treat mammals, including humans, suffering from bone-related disorders such as skeletal dysplasia. Non-limiting examples of CNP-responsive bone-related disorders and skeletal dysplasia include achondroplasia, decreased cartilage formation, short stature, dwarfism, osteochondrodysplasia, lethal achondroplasia, congenital osteogenic dysplasia, chondrodysplasia, congenital chondrodysplasia, homozygous achondroplasia, brachydactyly, congenital lethal hypophosphatase syndrome, perinatal lethal congenital osteogenic dysplasia, short rib polydactyly syndrome, limb root type congenital chondrodysplasia, Janssen type metaphyseal dysplasia, congenital The following are considered conditions: spinal epiphyseal dysplasia, osteodystrophy, malformed dysplasia, congenital short femur, Langer type midlimb dysplasia, Nivig type midlimb dysplasia, Robinnoc syndrome, Reinhardt syndrome, acrodysplasia, peripheral bone dysplasia, Knifell's dysplasia, fibrocartilage hyperplasia, Roberts syndrome, acromegaly, microlimb syndrome, Moquer syndrome, Knifell's syndrome, varicocele, and vertebral epiphyseal and metaphyseal dysplasia, as well as osteoporosis.

[0237] Other short stature and growth plate disorders covered by the method include those associated with mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, or FGFR3.

[0238] Other short stature and growth plate disorders covered by the method include those associated with mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, FGFR3, or IGF1R.

[0239] This article also provides treatments for short stature and growth plate disorders, including those associated with mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), SHOX, PTPN11, NPR2, NPPC, FGFR3, or IGF1R.

[0240] In addition, CNP variants can be used as adjuncts or alternatives to growth hormone for the treatment of idiopathic short stature and other skeletal dysplasia.

[0241] Growth plate disorders include conditions that cause short stature or abnormal bone growth and may be caused by gene mutations in genes involved in bone growth, including collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, or FGFR3. In various embodiments, growth plate disorders include conditions that cause short stature or abnormal bone growth and may be caused by gene mutations in genes involved in bone growth, including collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, FGFR3, or IGF1R. In various embodiments, growth plate disorders or short stature are associated with one or more mutations in RAS proteinopathy-associated genes. In various embodiments, growth plate disorders or short stature are associated with one or more mutations in the SHOX gene. In various embodiments, the mutations in growth plate genes in subjects with growth plate disorders are heterozygous. In various embodiments, the mutations are loss-of-function mutations. In various embodiments, the mutations are gain-of-function mutations. Growth plate disorders include, but are not limited to, familial short stature, dominant familial short stature (also known as dominant hereditary short stature), or idiopathic short stature. See, for example, Plachy et al., *Journal of Clinical Endocrinology and Metabolism* 104: 4273-4281, 2019.

[0242] Mutations in ACAN can cause familial osteochondritis dissecans and short stature, eventually leading to osteoarthritis, characterized by areas of bone damage (or lesions) caused by cartilage and sometimes bone detaching from the ends of the bone at the joint. It has been proposed that the lack of tissue in the cartilaginous network within growing bone can impair its growth, resulting in short stature. Mutations associated with ACAN and short stature include Val2303Met. See Stattin et al., *American Journal of Human Genetics* 86(2): 126-37, 2010. It is considered that patients with short stature due to ACAN mutations would benefit from CNP treatment, as CNP administration can increase the height of these patients through the known interaction between CNP and FGFR3.

[0243] Studies have shown that the natriuretic peptide system, including the receptor NPR2, is involved in the regulation of intrachondral bone growth (Vasquez et al., *Horm Res Pediat* 82:222-229, 2014). Research indicates that homozygous or compound heterozygous loss-of-function mutations in NPR2 cause Maroteaux-type acromegaly dysplasia (AMDM), a skeletal dysplasia characterized by extremely short stature (Vasquez et al., 2014, see above). Reports suggest that heterozygous loss-of-function (e.g., dominant-negative) NPR2 mutations are a cause of short stature, while gain-of-function heterozygous NPR2 mutations have been found to be a cause of tall stature (Vasquez et al., 2014, see above). Given that the interaction between CNP and NPR2 stimulates cGMP production, increasing cGMP levels is desirable in these cases and will have therapeutic benefits in the management of complications arising from these diseases and conditions.

[0244] Heterozygous mutations in NPR2 are thought to cause idiopathic short stature and other forms of short stature. Mutations in the NPR2 gene are described below and in Amano et al., *Journal of Clinical Endocrinology and Metabolism* 99:713-718, 2014; Hisado-Oliva et al., *Journal of Clinical Endocrinology and Metabolism* 100 1133-1 142, 2015; and Vasques et al., *Journal of Clinical Endocrinology and Metabolism* 98: E1636-1644, 2013, which are hereby incorporated by reference. Consideration is given to subjects with short stature who wish to be treated with the CNP variants described herein, having a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, and at least one of their parents having a height SDS of less than -1.0, -1.5, -2.0, or -2.5, optionally with the other parent having a height within the normal range. In various embodiments, the CNP variants can be used to treat subjects with short stature whose height SDS is between -2.0 and -3.0. In various embodiments, the CNP variants can be used to treat subjects with short stature whose height SDS is between -2.0 and -2.5. However, because de novo mutations in NPR2 cause short stature as defined by a height SDS of less than -1.5, -2.0, -2.5, or -3.0, treatment is also covered for individuals who are heterozygous carriers of a detrimental mutation in NPR2 and whose parents do not have short stature. Additionally, it covers the use of CNP treatment for individuals who are heterozygous for harmful mutations in other growth plate genes, in order to improve body size and / or enhance bone growth.

[0245] Exemplary NPR2 mutations in patients who can be treated with CNP variants include:

[0246]

[0247]

[0248] The role of NPPC in bone growth is well-established (Hisado-Oliva et al., *Genetics Medicine* 20:91-97, 2018). NPPC knockout mice exhibit severe disproportionate forms of dwarfism, including short limbs and endochondral ossification (Hisado-Oliva et al., 2018, see above). Human genome-wide studies have demonstrated an association between NPPC and height (Hisado-Oliva et al., 2018, see above). Although haploinous CNP deficiency is considered the cause of short stature in humans, recent studies have found heterozygous mutations in families with both short stature and short hands (Hisado-Oliva et al., 2018, see above). These studies observed a significant reduction in cGMP production measured in the heterozygous state (Hisado-Oliva et al., 2018, see above). Mutations in NPPC include the 355G>T missense mutation causing Gly119Cys changes and the 349C>G missense mutation causing Arg117Gly changes. Rescuing CGMP-derived CNP variants can provide therapeutic benefits in the management of patients with heterozygous loss-of-function NPPC mutations.

[0249] Isolated SHOX deficiency is one of the more common single-gene causes of short stature (birth incidence of approximately 10 / 100,000). [Marchini et al., Endocrine Review 37: 417-448, 2016] (Genoni 2018). The SHOX gene, located on both the X and Y chromosomes, encodes a transcription factor expressed throughout the growth plate that functions to influence the receptor B-type natriuretic peptide (NPR-B) and fibroblast growth factor receptor 3 (FGFR3) pathways. The SHOX gene has been shown to be a transcriptional repressor of FGFR3, and SHOX deficiency leads to increased FGFR3 signaling (Marchini, see above). Several pathogenic variants of SHOX are known to cause short stature. Individuals with pathogenic variants causing SHOX deficiency experience growth retardation in the first year of life, typically maintaining a height below -2.00 SDs compared to the CDC population standard, and do not develop a pubertal growth spurt, exacerbating the final size impairment (Binder 2018; Fukami 2016; Jorge 2010).

[0250] Turner syndrome is a rare chromosomal disorder that causes short stature and other phenotypic characteristics in girls (incidence rate of 32 per 100,000 females [16 per 100,000 total population] [Martin-Giacalone 2023]). It is caused by a structural abnormality or partial or complete deletion of one X chromosome, which in turn results in the loss of one copy of the SHOX gene (located on the X chromosome). The deletion of one copy of the SHOX gene causes growth disorders and skeletal abnormalities (Gravholt 2019). Girls with Turner syndrome exhibit a slowed growth rate after the first three years of their lives, and adult height is further affected by the absence of the pubertal growth spurt. Individuals with this syndrome are significantly shorter than average for females, with an average height difference of 20 cm from the median predicted height of their parents (Karlberg 1991; Rongen-Westerlaken 1997). Given the association of SHOX with FGFR3 and bone growth, subjects with homozygous or heterozygous SHOX mutations are considered to benefit from treatment with the CNP variants described herein.

[0251] Leri-Weill dyschondrosteosis (LWD) is a rare genetic disorder characterized by short forearms and lower legs, abnormal wrist misalignment (Madelung deformity), and short stature. LWD is caused by heterozygous mutations in the short stature homeobox (SHOX) gene or its regulatory elements located in pseudoautosomal region 1 (PAR1) of the sex chromosomes. (See Rare Disease Database; and Carmona et al., Human Molecular Genetics 20:1547-1559, 2011). The condition, Langer limb dysplasia, occurs in the presence of two SHOX mutations and can be caused by mutations on each chromosome (homozygous or compound heterozygous mutations). A subset of SHOX mutations causes idiopathic short stature. Turner syndrome occurs due to deletions on the X chromosome that may include the SHOX gene. SHOX has been identified as involved in FGFR3 transcriptional regulation and contributes to bone growth control (Marchini et al., *Endocrine Reviews* 37: 417-448, 2016). SHOX deficiency leads to increased FGFR3 signaling, and there is some evidence that SHOX also interacts directly with CNP / NPR2 (Marchini, see above). Given the association of SHOX with FGFR3 and bone growth, it is considered that subjects with homozygous or heterozygous SHOX mutations would benefit from treatment with the CNP variants described in this article.

[0252] Ras arthritis-associated protein kinase (RAS) disorders are a group of rare genetic disorders caused by mutations in genes involved in the Ras / mitogen-activated protein kinase (MAPK) pathway. RAS disorders are characterized by increased signaling via the RAS / MAPK pathway, which leads to downstream activation of the RAF / MEK / ERK pathway. Short stature is a characteristic feature of some RAS disorders. For example, CNP signaling inhibits RAF and reduces MEK and ERK activation.

[0253] This article covers the treatment of RAS protein disorders. RAS protein disorders associated with short stature include Noonan syndrome, Kestilo syndrome, cardiofacial skin syndrome, neurofibromatosis type 1, and leopard spot syndrome. Hereditary gingival fibromatosis type 1 is also a RAS protein disorder covered in this article. RAS protein disorders (including Noonan syndrome, Kestilo syndrome, cardiofacial skin syndrome, neurofibromatosis type 1, leopard spot syndrome, and hereditary gingival fibromatosis type 1) include patients with heterozygous variants of one or more of the following genes: BRAF, CBL, HRAS, KRAS, LZTR1, MAP2K1, MAP2K2, MRAS, NF1, NRAS, PPP1CB, PTPN11, RAF1, RRAS, RIT1, SHOC2, SOS1, or SOS2 (Tajan et al., Endocrine Review 2018;39(5):676-700).

[0254] CFC is caused by mutations in several genes, including K-Ras, B-Raf, Mek1, and Mek2, in the Ras / MAPK signaling pathway. Kristillo's syndrome (also known as facial cutaneous-bone (FCS) syndrome) is caused by an activating mutation in the H-Ras gene. Hereditary type I gingival fibroma (HGF) is caused by a dominant mutation in the Son of Sevenlesshomolog 1 (SOS1) gene, which encodes the guanine nucleotide exchange factor (SOS) of the Ras family, acting on small GTPases. Type I neurofibroma (NF1) is caused by mutations in the neurofibroma protein 1 gene, which encodes a negative regulator of the Ras / MAPK signaling pathway. Noonan's syndrome (NS) is caused by mutations in one of several genes, including PTPN1 1 (which encodes SHP2) and SOS1, as well as K-Ras and Raf-1.

[0255] CNP has been demonstrated to be an effective treatment in RAS protein disease models. Ono et al. induced mice lacking Nf1 in type I collagen-producing cells (Ono et al., Human Molecular Genetics 2013;22(15):3048-62). These mice exhibited constitutive ERK1 / 2 activation, as well as reduced chondrocyte proliferation and maturation. Daily injection of CNP into these mice reduced ERK phosphorylation and corrected for short stature. A mouse model of heart-face skin syndrome using a Braf mutant (p.Q241 R) (Inoue et al., Human Molecular Genetics 2019;28(1):74-83) showed reduced body length and growth plate width compared to wild type, as well as smaller proliferation and hypertrophic areas, and CNP administration increased body length in these animals.

[0256] Mutations in multiple genes can cause Noonan syndrome, characterized by short stature, heart defects, bleeding problems, and skeletal deformities. Mutations in the PTPN11 gene cause approximately half of all Noonan syndrome cases. Mutations in the SOS1 gene cause another 10 to 15% of cases, and mutations in the RAF1 and RIT1 genes each account for approximately 5% of cases. Mutations in other genes each account for a minority of cases. The cause of Noonan syndrome is unknown in 15 to 20% of the population with this condition.

[0257] Noonan syndrome (birth incidence 40 / 100,000 [NORD 2019]) is the most common RAS protein disorder. It is a group of clinically determined conditions caused by germline mutations in one of the genes encoding components of the RAS-MAPK pathway, which typically lead to increased signaling through this pathway. The RAS-MAPK pathway results in downstream activation of RAF / MEK / extracellular signal-regulated kinase (ERK). C-type natriuretic peptide (CNP) signaling interacts with this pathway by inhibiting RAF, resulting in reduced MEK and ERK activation. Noonan syndrome is primarily caused by gain-of-function (GoF) mutations in genes encoding components of the RAS-MAPK pathway. Noonan syndrome is characterized by short stature in 50–70% of patients (Bhambhani 2014), typical facial features and cardiac defects in over 80% of patients (Noonan 2005), and multisystem involvement in older children (Allanson 2021; Breilyn 2019). Growth retardation occurs in the first year of life, and children's height typically remains below -2.00 SDs until puberty, when growth is further affected by a weakened pubertal growth spurt (Carcavilla 2020). Approximately 50% of adults with Noonan syndrome have significantly reduced height (Noonan 2003).

[0258] The genes PTPN11, SOS1, RAF1, and RIT1 all encode important proteins in the RAS / MAPK cell signaling pathway, which is essential for cell division and growth (proliferation), differentiation, and cell migration. Many mutations in genes associated with Noonan syndrome enable the activation of these proteins, and this prolonged activation alters normal RAS / MAPK signaling, thereby disrupting the regulation of cell growth and division, leading to the characteristic features of Noonan syndrome. See, for example, Chen et al., Proceedings of the National Academy of Sciences 111(31):1 1473-8, 2014; Romano et al., Pediatrics 126(4): 746-59, 2010; Milosavljevic et al., American Journal of Medical Genetics 170(7): 1874-80, 2016. Subjects with mutations activating the MAPK pathway are considered to benefit from the CNP variant therapy described herein to improve bone growth and short stature. Additionally, subjects with mutations activating the MAPK pathway are considered to benefit from the CNP variant therapy described herein to improve other comorbidities associated with overactivated MAPK pathways in other cells throughout the body that express the NPR2 receptor on their surface.

[0259] Mutations in the PTPN11 gene, which encodes the non-receptor protein tyrosine phosphatase SHP-2, result in conditions characterized by short stature, such as Noonan syndrome (Musente et al., *Eur J HumGenet* 11:201-206 (2003)). Musente (see above) identified numerous mutations in the PTPN11 gene that contribute to short stature. Gain-of-function mutations lead to overactivation of signaling via SHP2 and inhibition of growth hormone-induced IGF-1 release, thereby contributing to slowed bone growth (Rocca Serra-Nedelec, *PNAS* 109:4257-4262, 2012). Subjects with homozygous or heterozygous PTPN11 mutations are considered to benefit from treatment with the CNP variants described herein to improve bone growth and short stature.

[0260] Mutations in the Indian hedgehog factor (IHH) gene, which is involved in the regulation of endochondral ossification, are also associated with short stature syndrome (Vasques et al., *Journal of Clinical Endocrinology and Metabolism* 103:604-614, 2018). Many of the identified IHH mutations are isolated from short stature in a dominant inheritance pattern. Given the association of IHH with bone growth and ossification, subjects with homozygous or heterozygous IHH mutations are considered to benefit from treatment with the CNP variants described in this article.

[0261] Mutations in FGFR3, including N540K and K650N, lead to short stature and reduced cartilage production.

[0262] Insulin-like growth factor 1 receptor (IGF1R) is a heterotetrameric (α2β2) transmembrane glycoprotein with intrinsic kinase activity. IGF1R has been shown to play a role in prenatal and postnatal growth. Heterozygous mutations in IGF1R have been identified in small forgestational age (SGA) infants and individuals with familial short stature (Kawashima et al., *Endocrine J* 59:179-185, 2012). Mutations in IGF1R associated with short stature include R108Q / K115N, R59T, R709Q, G1050K, R481Q, V599E, and G1125A (Kawashima, see above).

[0263] Height is a highly heritable trait, potentially influenced by the combined effects of hundreds or thousands of genes (Wood et al., 2014, *Nature Genetics*, 46:1 173-1 189). Short stature in individuals is likely the result of the combined effects of these genes, rather than a single gene being the primary contributing factor. Given that CNPs can increase length in normal animals, such as by enhancing bone growth and length, it is considered that individuals with short stature could potentially benefit from treatment with CNP variants defined as a height SDS less than -1.0, -1.5, -2.0, -2.5, or -3.0.

[0264] In various embodiments, CNP variants can be used to treat subjects with short stature whose height SDS is less than -1.0, -1.5, -2.0, -2.5, or -3.0, and at least one of their parents has a height SDS less than -1.0, -1.5, -2.0, or -2.5, optionally wherein the other parent's height is within the normal range. In various embodiments, CNP variants can be used to treat subjects with short stature whose height SDS is between -2.0 and -3.0. In various embodiments, CNP variants can be used to treat subjects with short stature whose height SDS is between -2.0 and -2.5. In various embodiments, CNP variants can be used to treat subjects with short stature whose height SDS is -2.00 or lower. In various embodiments, short stature is associated with mutations in one or more genes associated with short stature, such as collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, FGFR3, SHOX, or insulin-like growth factor 1 receptor (IGF1R), or combinations thereof. In various embodiments, mutations in the toothless E3 ubiquitin protein ligase homolog (DTL) and pregnancy-associated plasma protein A2 (PAPPA2), or combinations thereof, are also associated with short stature.

[0265] In various embodiments, growth plate syndrome or short stature is associated with one or more mutations in RAS protein disease-related genes.

[0266] In various embodiments, short stature was determined to be the result of mutations in multiple genes using a polygenic risk score (PRS). The polygenic risk score (PRS) for height was calculated using the disclosed maximum GWAS comprehensive analysis for height, which did not include any samples from the UK Biobank project as described in Example 4. The cohort was divided into five PRS quintiles (PRS 1 being the shortest height and PRS 5 being the tallest). In various embodiments, subjects had an NPR2 mutation and low PRS. In various embodiments, subjects had an FGFR3 mutation and low PRS. In various embodiments, subjects had an NPR2 mutation and low PRS. In various embodiments, subjects had an IGF1R mutation and low PRS. In various embodiments, subjects had an NPPC mutation and low PRS. In various embodiments, subjects had a SHOX mutation and low PRS. In various embodiments, subjects had one or more mutations in one or more of FGFR3, IGF1R, NPPC, NPR2, and SHOX, and low PRS. In various embodiments, the subject has one or more mutations in the toothless E3 ubiquitin protein ligase homolog (DTL) or pregnancy-associated plasma protein A2 (PAPPA2). In various embodiments, the PRS score is 1 or 2. In various embodiments, the PRS score is 1. In various embodiments, the PRS score is 2.

[0267] In addition, CNP variants can be used to treat other bone-related conditions and disorders, such as rickets, hypophosphatemic rickets [including X-linked hypophosphatemic rickets (also known as vitamin D-resistant rickets) and autosomal dominant hypophosphatemic rickets], and osteomalacia [including tumor-induced osteomalacia (also known as carcinogenic osteomalacia or carcinogenic hypophosphatemic osteomalacia)].

[0268] The CNP variant disclosed herein can also be used to treat osteoarthritis. Osteoarthritis is a degenerative disease of articular cartilage and occurs frequently in older adults. Osteoarthritis involves cartilage destruction and proliferative changes in bone and cartilage caused by degeneration of joint components, which lead to secondary arthritis (e.g., synovitis). In osteoarthritis, extracellular matrix proteins, which are the functional entities of cartilage, are reduced, and the number of chondrocytes is reduced (Arthritis and Rheumatology (Arth. Rheum.) 46(8): 1986-1996 (2002)). By promoting matrix production, growth, and differentiation of chondrocytes, the CNP composition can be used to counteract unwanted FGF-2 effects and increase matrix synthesis in subjects suffering from arthritis, including osteoarthritis, thereby treating arthritis, including osteoarthritis.

[0269] In some embodiments, the CNP variants of this disclosure, as well as compositions and formulations comprising CNP variants, can be used to improve one or more symptoms or physiological consequences of skeletal dysplasia, wherein said improvement may be an increase in absolute growth, an increase in growth rate, an increase in quantitative computed tomography (QCT) bone mineral density, an improvement in growth plate morphology, an increase in long bone growth, an improvement in spinal morphology, an improvement in elbow range of motion, and / or a reduction in sleep apnea. Other symptoms that can be improved by CNP therapy include tissue mineral density (TMD), bone mineral density (BMD), bone strength, length of metacarpals with a greater cortical area, or combinations thereof. In this regard, it should be noted that the terms “improved,” “improvement,” “increase,” “decrease,” and their grammatical equivalents, when used with respect to the symptoms or physiological consequences of a disease condition, are relative terms, meaning a comparison of the state of the symptoms or physiological consequences of the disease after treatment with the CNP variants of the present invention (or compositions or formulations comprising said CNP variants) to the state of the same symptoms or physiological consequences of the disease before treatment with the CNP variants of the present invention (or compositions or formulations comprising said CNP variants) (i.e., a comparison with a “baseline”). As described above, the “baseline” status can be determined by measuring the status of a subject before treatment (which can then be compared with the status of the same subject after treatment), or by measuring the status of a group of subjects suffering from the same ailment with the same or similar characteristics (e.g., age, sex, and / or disease condition or progression).

[0270] Also provided is a method for overcoming cell growth arrest induced by constitutively active mutant fibroblast growth factor receptor 3 (FGFR-3), the method comprising contacting cells expressing the constitutively active FGFR-3 with the CNP variant or composition described herein.

[0271] Another method is provided for stimulating cGMP production in cells expressing natriuretic natriuretic peptide receptor B (NPR-B), the method comprising contacting NPR-B-expressing cells with a CNP variant or composition described herein.

[0272] On the other hand, this article provides a method for increasing facial volume, facial sinus volume, and foramen magnum area in subjects (e.g., 6 months of age or younger) with bone-related disorders, skeletal dysplasia, or short stature, the method comprising administering a CNP variant described herein. A method for reducing the incidence of sudden infant death syndrome, sleep apnea, and the necessity of neurosurgical decompression of the foramen magnum in subjects (e.g., 6 months of age or younger) with bone-related disorders, skeletal dysplasia, or short stature is also provided, the method comprising administering a CNP variant described herein.

[0273] In various embodiments, increases in facial volume, facial sinus volume, and foramen magnum area were measured using magnetic resonance imaging (MRI). In various embodiments, changes in facial volume, facial sinus volume, and foramen magnum area were compared to baseline levels, healthy controls, or untreated controls.

[0274] In yet another embodiment, this disclosure provides CNP variants that, upon in vitro or in vivo stimulation, produce at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the cGMP levels produced at the same wt CNP22 concentration (e.g., 1 μM). In yet another embodiment, the CNP variants of this disclosure, upon in vitro or in vivo stimulation, produce at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the cGMP levels produced at the same wt CNP22 concentration (e.g., 1 μM).

[0275] In consideration, any of the CNP variants described herein can be used in the method.

[0276] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37) (SEQ ID NO: 1).

[0277] In various embodiments, the peptide further comprises an acetyl group. In various embodiments, the acetyl group is located at the N-terminus of the peptide. In various embodiments, the peptide further comprises an OH or NH2 group at the C-terminus. In various embodiments, the variant comprises one or more linker groups described herein. In various embodiments, the linker is a hydrolyzable linker.

[0278] Treatment efficacy was measured using various parameters. In various embodiments, efficacy was assessed as the change in annualized growth rate from baseline to the intervention period. Efficacy was also assessed as the change in height SDS from baseline to the end of treatment, measured using CDC growth curves, and the growth rate SDS was based on bone mineral density in pediatric studies (Kelly et al., Journal of Clinical Endocrinology and Metabolism 2014;99(6):2104-2112).

[0279] QoLISSY, or Quality of Life in Short Stature Children, is a guided assessment (see *Quality of Life in Short Stature Youth - The QoLISSY Questionnaire User's Manual*, Lengerich: Pabst Science Publishers; 2013). The QoLISSY questionnaire is a disease-specific clinical outcome assessment designed for children with short stature. It has both child-reported and parent-reported versions and consists of questions in eight domains: physical, social, emotional, coping, dealing, beliefs, future, and impact on parents. The total scores for each domain are then linearly transformed into a standardized score on a 0-100 scale: lowest score = 0 (worst QoL), highest score = 100 (best QoL).

[0280] In various embodiments, subjects receiving the CNP prodrug described herein to treat skeletal dysplasia had reduced or no cardiovascular (CV) side effects, such as changes in systemic blood pressure (mean arterial pressure, systolic and diastolic blood pressure, pulse pressure) and heart rate, as observed with the administration of non-prodrug CNP variants.

[0281] Pharmaceutical Composition

[0282] This disclosure provides pharmaceutical compositions, including modulated-release compositions, which comprise CNP variants described herein, and one or more pharmaceutically acceptable excipients, carriers, and / or diluents. In some embodiments, the compositions further comprise one or more other bioactive agents (e.g., proteases, receptor tyrosine kinases, and / or inhibitors that clear receptor NPR-C).

[0283] This disclosure provides modulated release compositions comprising the conjugated portions described herein. Modulated release compositions include those that deliver a drug at a delayed time after administration (delayed-release dose) or deliver a drug over an extended period of time (extended-release dose). Various embodiments of CNP peptide conjugates provided herein include modulated release compositions, such as extended-release, sustained-release, or controlled-release, as well as delayed-release compositions. The term "extended-release composition" refers to a composition formulated in a manner that facilitates the preparation of an active ingredient / drug available for use over an extended period after administration (US Pharmacopeia). Extended-release doses include sustained-release (SR) or controlled-release (CR) forms, wherein sustained-release maintains the duration of drug release but not necessarily at a constant rate, while CR maintains the duration of drug release at a nearly constant rate (Pharmaceutics: Drug Delivery and Targeting, Yvonne Perrie, Thomas Rades, Pharmaceutical Press, 2009). Delayed-release compositions or products are modified to delay the release of the active pharmaceutical ingredient over a period of time following initial administration.

[0284] In various embodiments, the modulated composition is a prolonged release composition.

[0285] In various embodiments, for the extended release composition, at pH 7 to 7.6, (i) less than about 20% of the peptide is released on day 1; and (ii) about 90% of the peptide is released weekly, or about 90% of the peptide is released every two weeks, or about 90% of the peptide is released monthly.

[0286] In various embodiments, less than about 20% of the peptide is released on day 1 at pH 7 to 7.6. In various embodiments, less than about 10% of the peptide is released on day 1 at pH 7 to 7.6. Further consideration was given to (i) less than about 30%, or about 40%, or about 50% of the peptide being released on day 1 at pH 7.0 to 7.6; and (ii) about 90% of the peptide being released weekly, or bi-weekly, or monthly at pH 7 to 7.6. Further consideration was given to the following: (i) at pH 7.0 to 7.6, less than about 30%, or about 40%, or about 50%, or about 60% of the peptides were released on day 1; and (ii) at pH 7 to 7.6, about 70%, about 80%, or about 90% of the peptides were released weekly; or about 70%, about 80%, or about 90% of the peptides were released every two weeks; or about 70%, about 80%, or about 90% of the peptides were released every three weeks; or about 70%, about 80%, or about 90% of the peptides were released monthly. In various embodiments, about 90% of the peptides were released weekly at pH 7 to 7.6. In various embodiments, about 90% of the peptides were released every two weeks at pH 7 to 7.6. In various embodiments, about 90% of the peptides were released monthly at pH 7 to 7.6. Upon further consideration, the release may be carried out at pH 7.0 to 7.6, pH 7.1 to 7.5, pH 7.2 to 7.4, pH 7.2 to 7.6, or pH 7.0 to 7.4.

[0287] In various embodiments, (i) less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the peptide is released on day 1 at pH 7.0 to 7.6; and (ii) about 90% of the peptide is released weekly, or bi-weekly, or monthly at pH 7 to 7.6. Upon further consideration, (i) at pH 7.0 to 7.6, less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the peptides are released on day 1; and (ii) at pH 7 to 7.6, about 70%, about 80%, or about 90% of the peptides are released weekly; or about 70%, about 80%, or about 90% of the peptides are released every two weeks; or about 70%, about 80%, or about 90% of the peptides are released every three weeks; or about 70%, about 80%, or about 90% of the peptides are released monthly; or (ii) at pH At levels 7 to 7.6, approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released weekly; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released every two weeks; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released every three weeks; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released monthly.

[0288] In various embodiments, the composition comprises an excipient, a diluent, or a carrier. In various embodiments, the extended-release composition comprises an excipient, a diluent, or a carrier. In various embodiments, the excipient, diluent, or carrier is a pharmaceutically acceptable excipient, diluent, or carrier.

[0289] Non-limiting examples of excipients, carriers, and diluents include mediators, liquids, buffers, isotonic agents, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, adjuvants, etc. The composition may contain liquids (e.g., water, ethanol); diluents having various buffer contents (e.g., Tris-HCl, phosphate, acetate buffers, citrate buffers), pH, and ionic strength; detergents and solubilizers (e.g., polysorbate 20, polysorbate 80); antioxidants (e.g., methionine, ascorbic acid, sodium metabisulfite); preservatives (e.g., thimerosal, benzyl alcohol, m-cresol); and additives (e.g., lactose, mannitol, sucrose). The use of excipients, diluents and carriers in the formulation of pharmaceutical compositions is known in the art; see, for example, Remington Pharmaceutical Science, 18th edition, pp. 1435-1712, Mack Publishing Co. (Easton, Pennsylvania (1990)), which is incorporated herein by reference in its entirety.

[0290] For example, carriers include, but are not limited to, diluents, mediators, and adjuvants, as well as implant carriers, and inert, non-toxic solid or liquid fillers and encapsulating materials that do not react with one or more active ingredients. Non-limiting examples of carriers include phosphate-buffered saline, physiological saline, water, and emulsions (e.g., oil / water emulsions). Carriers can be solvents or dispersion media containing, for example, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, and mixtures thereof. In some embodiments, the composition is a liquid formulation. In various embodiments, the CNP variant is reconstituted from a lyophilized powder. In some embodiments, the formulation comprises a CNP variant at a concentration ranging from about 0.1 mg / ml to about 20 mg / ml, or from about 0.5 mg / ml to about 20 mg / ml, or from about 1 mg / ml to about 20 mg / ml, or from about 0.1 mg / ml to about 10 mg / ml, or from about 0.5 mg / ml to about 10 mg / ml, or from about 0.5 to 5 mg / ml, or from about 0.5 to 3 mg / ml, or from about 1 mg / ml to about 10 mg / ml. In various embodiments, the concentration of the CNP variant is from 0.8 mg / ml to 2 mg / ml. In various embodiments, the concentration of the CNP variant is 0.8 mg / ml. In various embodiments, the formulation comprises a CNP variant concentration of not less than about 10 mg / ml, not less than about 5 mg / ml, not less than about 1 mg / ml, not less than about 0.5 mg / ml, or not less than about 0.1 mg / ml. In various embodiments, the formulation comprises a CNP variant concentration not exceeding about 300 mg / ml, not exceeding about 250 mg / ml, not exceeding about 200 mg / ml, not exceeding about 150 mg / ml, not exceeding about 100 mg / ml, not exceeding about 50 mg / ml, or not exceeding about 40 mg / ml. In some embodiments, the formulation comprises a CNP variant at concentrations ranging from about 1 mg / ml to about 300 mg / ml, or about 5 mg / ml to about 300 mg / ml, or about 10 mg / ml to about 300 mg / ml, or about 1 mg / ml to about 150 mg / ml, or about 1 mg / ml to about 75 mg / ml, or about 1 to 75 mg / ml, or about 5 to 50 mg / ml. In various embodiments, the concentration of the CNP variant is from 10 mg / ml to 30 mg / ml. In various embodiments, the concentration of the CNP variant is 10 mg / ml. In various embodiments, the concentration of the CNP variant is 30 mg / ml. In other embodiments, the formulation comprises a concentration ranging from about 0.1 mg / ml to about 20 mg / ml, or about 0.5 mg / ml to about 20 mg / ml, or about 1 mg / ml to about 20 mg / ml, or about 0.1 mg / ml to about 10 mg / ml, or about 0.5 mg / ml...CNP variants at concentrations of approximately 10 mg / ml to about 10 mg / ml, or approximately 0.5 to 5 mg / ml, or approximately 0.5 to 3 mg / ml, or approximately 1 mg / ml to about 10 mg / ml. In various embodiments, the concentration of the CNP variant is 0.8 mg / ml to 2 mg / ml. In various embodiments, the concentration of the CNP variant is 0.8 mg / ml. In various embodiments, the concentration of the CNP variant is 2.0 mg / ml. In various embodiments, the concentration of the CNP variant is 10 mg / ml. Exemplary concentrations of CNP variants in formulations are 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 49 mg / ml, 50 mg / ml, 51 mg / ml, 52 mg / ml, 53 mg / ml, 54 mg / ml, 55 mg / ml, 56 mg / ml, 57 mg / ml, 58 mg / ml, 59 mg / ml, 60 mg / ml, 61 mg / ml, 62 mg / ml, 63 mg / ml, 64 mg / ml, 65 mg / ml, 66 mg / ml, 67 mg / ml, 68 mg / ml, 69 mg / ml, 70mg / ml, 71 mg / ml, 72 mg / ml, 73 mg / ml, 74 mg / ml, 75 mg / ml, 76 mg / ml, 77 mg / ml, 78 mg / ml, 79 mg / ml, 80 mg / ml, 81mg / ml、82 mg / ml、83 mg / ml、84 mg / ml、85 mg / ml、86 mg / ml、87mg / ml、88 mg / ml、89 mg / ml、90 mg / ml、91 mg / ml、92 mg / ml、93 mg / ml、94 mg / ml、95 mg / ml、96 mg / ml、97 mg / ml、98 mg / ml、99 mg / ml、100 mg / ml、101 mg / ml、102 mg / ml、103 mg / ml、104 mg / ml、105 mg / ml、106 mg / ml、107 mg / ml、108 mg / ml、109 mg / ml、110 mg / ml、111mg / ml、112 mg / ml、113 mg / ml、114 mg / ml、115 mg / ml、116 mg / ml、117 mg / ml、118 mg / ml、119 mg / ml、120 mg / ml、121 mg / ml、122 mg / ml、123 mg / ml、124 mg / ml、125 mg / ml、126 mg / ml、127 mg / ml、128 mg / ml、129 mg / ml、130 mg / ml、131 mg / ml、132 mg / ml、133 mg / ml、134mg / ml、135 mg / ml、136 mg / ml、137 mg / ml、138 mg / ml、139 mg / ml、140 mg / ml、141 mg / ml、142 mg / ml、143 mg / ml、144 mg / ml、145 mg / ml、146 mg / ml、147 mg / ml、148 mg / ml、149 mg / ml、150 mg / ml、151 mg / ml、152 mg / ml、153 mg / ml、154 mg / ml、155 mg / ml、156 mg / ml、157mg / ml、158 mg / ml、159 mg / ml、160 mg / ml、161 mg / ml、162 mg / ml、163 mg / ml、164 mg / ml、165 mg / ml、166 mg / ml、167 mg / ml、168 mg / ml、169 mg / ml、170 mg / ml、171 mg / ml、172 mg / ml、173 mg / ml、174 mg / ml、175 mg / ml、176 mg / ml、177 mg / ml、178 mg / ml、179 mg / ml、180mg / ml、181 mg / ml、182 mg / ml、183mg / ml、184 mg / ml、185 mg / ml、186 mg / ml、187 mg / ml、188 mg / ml、189 mg / ml、190 mg / ml、191 mg / ml、192 mg / ml、193 mg / ml、194 mg / ml、195 mg / ml、196 mg / ml、197 mg / ml、198 mg / ml、199 mg / ml、200 mg / ml、201 mg / ml、202 mg / ml、203mg / ml、204 mg / ml、205 mg / ml、206 mg / ml、207 mg / ml、208 mg / ml、209 mg / ml、210 mg / ml、211 mg / ml、212 mg / ml、213 mg / ml、214 mg / ml、215 mg / ml、216 mg / ml、217 mg / ml、218 mg / ml、219 mg / ml、220 mg / ml、221 mg / ml、222 mg / ml、223 mg / ml、224 mg / ml、225 mg / ml、226mg / ml、227 mg / ml、228 mg / ml、229 mg / ml、230 mg / ml、231 mg / ml、232 mg / ml、233 mg / ml、234 mg / ml、235 mg / ml、236 mg / ml、237 mg / ml、238 mg / ml、239 mg / ml、240 mg / ml、241 mg / ml、242 mg / ml、243 mg / ml、244 mg / ml、245 mg / ml、246 mg / ml、247 mg / ml、248 mg / ml、249mg / ml、250 mg / ml、251 mg / ml、252 mg / ml、253 mg / ml、254 mg / ml、255 mg / ml、256 mg / ml、257 mg / ml、258 mg / ml、259 mg / ml、260 mg / ml、261 mg / ml、262 mg / ml、263 mg / ml、264 mg / ml、265 mg / ml、266 mg / ml、267 mg / ml、268 mg / ml、269 mg / ml、270 mg / ml、271 mg / ml、272mg / ml、273 mg / ml、274 mg / ml、275 mg / ml、276 mg / ml、277 mg / ml、278 mg / ml、279 mg / ml、280 mg / ml、281 mg / ml、282 mg / ml、283mg / ml, 284 mg / ml, 285 mg / ml, 286 mg / ml, 287 mg / ml, 288 mg / ml, 289 mg / ml, 290 mg / ml, 291 mg / ml, 292 mg / ml, 293 mg / ml, 294 mg / ml, 295mg / ml, 296 mg / ml, 297 mg / ml, 298 mg / ml, 299 mg / ml or 300 mg / ml.

[0291] In other embodiments, the composition comprises a buffer solution or buffering agent to maintain the pH of the CNP-containing solution or suspension within a desired range. Non-limiting examples of buffer solutions include phosphate-buffered saline, Tris-buffered saline, and Hank's buffered saline. Buffering agents include, but are not limited to, sodium acetate, sodium phosphate, and sodium citrate. Mixtures of buffering agents may also be used. In some embodiments, the buffering agent is histidine / L-histidine or histidine monohydrochloride monohydrate. In other embodiments, the buffering agent is acetate / acetate or citric acid / citrate. The suitable amount of buffering agent in the composition depends in part on the specific buffering agent used and the desired pH of the solution or suspension. In some embodiments, the concentration of the buffering agent is about 10 mM ± 5 mM. In some embodiments, the pH of the composition is from about pH 3 to about pH 9, or from about pH 3 to about pH 7.5, or from about pH 3.5 to about pH 7, or from about pH 3.5 to about pH 6.5, or from about pH 4 to about pH 6, or from about pH 4 to about pH 5, or about pH 5.0 ± 1.0. In various embodiments, the pH is from about 5.0 to about 6.0 (e.g., 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0). In various embodiments, the pH is 5.5.

[0292] In other embodiments, the composition contains an isotonic adjuster to make the solution or suspension isotonic and more suitable for application. Non-limiting examples of isotonic agents include NaCl, dextrose, glucose, glycerol, sorbitol, xylitol, and ethanol. In some embodiments, the isotonic agent is NaCl. In some embodiments, the concentration of NaCl is about 160 ± 20 mM, or about 140 mM ± 20 mM, or about 120 ± 20 mM, or about 100 mM ± 20 mM, or about 80 mM ± 20 mM, or about 60 mM ± 20 mM.

[0293] In other embodiments, the composition comprises a preservative. Preservatives include, but are not limited to, m-cresol and benzyl alcohol. In some embodiments, the concentration of the preservative is about 0.4% ± 0.2%, or about 1% ± 0.5%, or about 1.5% ± 0.5%, or about 2.0% ± 0.5%.

[0294] In other embodiments, the composition contains an anti-adsorption agent (e.g., to reduce the adsorption of CNP variants onto glass or plastic). Anti-adsorption agents include, but are not limited to, benzyl alcohol, polysorbate 20, and polysorbate 80. In some embodiments, the concentration of the anti-adsorption agent is about 0.001% to about 0.5%, or about 0.01% to about 0.5%, or about 0.1% to about 1%, or about 0.5% to about 1%, or about 0.5% to about 1.5%, or about 0.5% to about 2%, or about 1% to about 2%.

[0295] In other embodiments, the composition comprises a stabilizer. Non-limiting examples of stabilizers include glycerol, glycerol, thioglycerol, methionine (L-methionine), and ascorbic acid and its salts. In some embodiments, when the stabilizer is thioglycerol or ascorbic acid or its salts, the concentration of the stabilizer is from about 0.1% to about 1%. In other embodiments, when the stabilizer is methionine, the concentration of the stabilizer is from about 0.01% to about 0.5%, or from about 0.01% to about 0.2%. In still other embodiments, when the stabilizer is glycerol, the concentration of the stabilizer is from about 5% to about 100% (pure).

[0296] In other embodiments, the composition contains an antioxidant. Exemplary antioxidants include, but are not limited to, methionine and ascorbic acid. In some embodiments, the molar ratio of the antioxidant to CNP is about 0.1:1 to about 15:1, or about 1:1 to about 15:1, or about 0.5:1 to about 10:1, or about 1:1 to about 10:1, or about 3:1 to about 10:1.

[0297] Pharmaceutically acceptable salts may be used in the compositions, including but not limited to inorganic acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate), organic acid salts (e.g., acetate, propionate, malonate, benzoate, methanesulfonate, toluenesulfonate), and amine salts (e.g., isopropylamine, trimethylamine, dicyclohexylamine, diethanolamine). A detailed discussion of pharmaceutically acceptable salts can be found in Remington Pharmaceutical Science, 18th edition, Mack Publishing Company (Easton, Pennsylvania (1990)).

[0298] Pharmaceutical compositions can be administered in various forms, such as tablets, capsules, granules, powders, solutions, suspensions, emulsions, ointments, and transdermal patches. The dosage form of the composition can be tailored to the desired mode of administration. For oral administration, the composition may be in the form of, for example, tablets or capsules (including soft gel capsules), or may be, for example, aqueous or non-aqueous solutions, suspensions, or syrups. Tablets and capsules for oral administration may include one or more common excipients, diluents, and carriers, such as mannitol, lactose, glucose, sucrose, starch, corn starch, sodium saccharin, talc, cellulose, magnesium carbonate, and lubricants (e.g., magnesium stearate, sodium stearoyl fumarate). If necessary, flavoring agents, coloring agents, and / or sweeteners may be added to solid and liquid formulations.

[0299] Other optional ingredients for oral formulations include, but are not limited to, preservatives, suspending agents, and thickeners. Oral formulations may also have an enteric coating to protect CNP variants from the acidic environment of the stomach. Methods for preparing solid and liquid dosage forms are known to or will be obvious to those skilled in the art (see, for example, Remington Pharmaceutical Sciences mentioned above).

[0300] Formulations intended for parenteral administration can be prepared, for example, in the form of a liquid solution or suspension, a solid form suitable for dissolving or suspending in a liquid medium prior to injection, or an emulsion. For example, sterile injectable solutions and suspensions can be formulated using suitable diluents, carriers, solvents (e.g., buffered aqueous solutions, Ringer's solution, isotonic sodium chloride solution), dispersants, wetting agents, emulsifiers, suspending agents, etc., according to techniques known in the art. Additionally, sterile non-volatile oils, fatty esters, polyols, and / or other inactive ingredients may be used. As another example, formulations intended for parenteral administration include aqueous sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners.

[0301] Compositions containing CNP variants may also be lyophilized formulations. In some embodiments, the lyophilized formulations contain buffers and build-up agents, and optionally antioxidants. Exemplary buffers include, but are not limited to, acetate buffers, citrate buffers, and histidine buffers. Exemplary build-up agents include, but are not limited to, mannitol (e.g., D-mannitol), sucrose, dextran, lactose, trehalose (e.g., trehalose dihydrate), and povidone (PVP K24). In some embodiments, the amount of mannitol is about 3% to about 10%, or about 4% to about 8%, or about 4% to about 6%. In some embodiments, the amount of sucrose is about 6% to about 20%, or about 6% to about 15%, or about 8% to about 12%. Exemplary antioxidants include, but are not limited to, methionine and ascorbic acid. In various embodiments, the lyophilized formulation comprises not less than about 10 mg of a CNP variant, not less than about 5 mg of a CNP variant, not less than about 3 mg of a CNP variant, not less than about 1 mg of a CNP variant, not less than about 0.5 mg of a CNP variant, or not less than about 0.1 mg of a CNP variant. In various embodiments, the lyophilized formulation comprises not more than about 300 mg of a CNP variant, not more than about 250 mg of a CNP variant, not more than about 200 mg of a CNP variant, not more than about 150 mg of a CNP variant, not more than about 100 mg of a CNP variant, not more than about 50 mg of a CNP variant, or not more than about 40 mg of a CNP variant. In various embodiments, the lyophilized formulation comprises about 1 mg to about 300 mg of a CNP variant, or about 5 mg to about 300 mg of a CNP variant, or about 10 mg to about 300 mg of a CNP variant, or about 1 mg to about 150 mg of a CNP variant, or about 5 mg to about 150 mg of a CNP variant, or about 5 mg to 150 mg of a CNP variant, or about 5 mg to 75 mg of a CNP variant, or about 10 mg to about 50 mg of a CNP variant. The lyophilized formulation comprises 13 mg to 39 mg of a CNP variant. In various embodiments, the lyophilized formulation comprises 13 mg of a CNP variant. In various embodiments, the lyophilized formulation comprises 39 mg of a CNP variant. In other embodiments, the lyophilized formulation comprises about 0.1 mg to about 20 mg of a CNP variant, or about 0.4 mg to about 20 mg of a CNP variant, or about 1 mg to about 20 mg of a CNP variant, or about 0.1 mg to about 10 mg of a CNP variant, or about 0.1 mg to about 10 mg of a CNP variant, or about 0.1 mg to 5 mg of a CNP variant. In various embodiments, the lyophilized formulation comprises 0.4 mg to 3.5 mg of a CNP variant. In various embodiments, the lyophilized formulation comprises 0.4 mg of a CNP variant. In various embodiments, the lyophilized formulation comprises 0.56 mg of the CNP variant. In various embodiments, the lyophilized formulation contains 1.2 mg of the CNP variant. In various embodiments, the lyophilized formulation contains 3.5 mg of the CNP variant. Exemplary concentrations of CNP variants in the formulation are 0.1 mg, 0.11 mg, 0.12 mg, 0.13 mg, 0.14 mg, 0.15 mg, 0.16 mg, 0.17 mg, 0.18 mg, 0.19 mg, 0.2 mg, 0.21 mg, 0.22 mg, 0.23 mg, 0.24 mg, 0.25 mg, 0.26 mg, 0.27 mg, 0.28 mg, 0.29 mg, 0.3 mg, 0.31 mg, 0.32 mg, 0.33 mg, 0.34 mg, 0.35 mg, 0.36 mg, 0.37 mg, 0.38 mg, 0.39 mg, 0.4 mg, 0.41 mg, 0.42 mg, 0.43 mg, 0.44 mg, 0.45 mg, 0.46 mg, 0.47 mg, 0.48 mg, and 0.49 mg. 0.5 mg mg, 0.69 mg, 0.7 mg, 0.71 mg, 0.72 mg, 0.73 mg, 0.74 mg, 0.75 mg, 0.76 mg, 0.77 mg, 0.78 mg, 0.79mg, 0.8 mg, 0.81 mg, 0.82 mg, 0.83 mg, 0.84 mg, 0.85 mg, 0.86 mg, 0.87 mg, 0.88 mg, 0.89 mg, 0.9 mg, 0.91 mg, 0.92 mg, 0.93 mg, 0.94 mg, 0.95 mg, 0.96 mg, 0.97 mg, 0.98mg, 0.99 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg, 13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg, 16.5 mg、17 mg、17.5mg、18 mg、18.5 mg、19 mg、19.5 mg、20 mg、20.5 mg、21 mg、21.5 mg、22 mg、22.5 mg、23mg、23.5 mg、24 mg、24.5 mg、25 mg、25.5 mg、26 mg、26.5 mg、27 mg、27.5 mg、28 mg、28.5mg、29 mg、29.5 mg、30 mg、30.5 mg、31 mg、31.5 mg、32 mg、32.5 mg、33 mg、33.5 mg、34mg、34.5 mg、35 mg、35.5 mg、36 mg、36.5 mg、37 mg、37.5 mg、38 mg、38.5 mg、39 mg、39.5mg、40 mg、40.5 mg、41 mg、41.5 mg、42 mg、42.5 mg、43 mg、43.5 mg、44 mg、44.5 mg、45mg、45.5 mg、46 mg、46.5 mg、47 mg、47.5 mg、48 mg、48.5 mg、49 mg、49.5 mg、50 mg、50.5mg、51 mg、51.5 mg、52 mg、52.5 mg、53 mg、53.5 mg、54 mg、54.5 mg、55 mg、55.5 mg、56mg、56.5 mg、57 mg、57.5 mg、58 mg、58.5 mg、59 mg、59.5 mg、60 mg、60.5 mg、61 mg、61.5mg、62 mg、62.5 mg、63 mg、63.5 mg、64 mg、64.5 mg、65 mg、65.5 mg、66 mg、66.5 mg、67mg、67.5 mg、68 mg、68.5 mg、69 mg、69.5 mg、70 mg、70.5 mg、71 mg、71.5 mg、72 mg、72.5mg、73 mg、73.5 mg、74 mg、74.5 mg、75 mg、75.5 mg、76 mg、76.5 mg、77 mg、77.5 mg、78mg、78.5 mg、79 mg、79.5 mg、80 mg、80.5 mg、81 mg、81.5 mg、82 mg、82.5 mg、83 mg、83.5mg、84 mg、84.5 mg、85 mg、85.5 mg、86 mg、86.5 mg、87 mg、87.5 mg、88 mg、88.5 mg、89mg、89.5 mg、90 mg、90.5 mg、91 mg、91.5 mg、92 mg、92.5 mg、93 mg、93.5 mg、94 mg、94.5mg、95 mg、95.5 mg、96 mg、96.5 mg、97 mg、97.5 mg、98 mg、98.5 mg、99 mg、99.5 mg、100mg、100.5 mg、101 mg、101.5 mg、102 mg、102.5 mg、103 mg、103.5 mg、104 mg、104.5 mg、105 mg、105.5 mg、106 mg、106.5 mg、107 mg、107.5 mg、108 mg、108.5 mg、109 mg、109.5mg、110 mg、110.5 mg、111 mg、111.5 mg、112 mg、112.5 mg、113 mg、113.5 mg、114 mg、114.5 mg、115 mg、115.5 mg、116 mg、116.5 mg、117 mg、117.5 mg、118 mg、118.5 mg、119mg、119.5 mg、120 mg、120.5 mg、121 mg、121.5 mg、122 mg、122.5 mg、123 mg、123.5 mg、124 mg、124.5 mg、125 mg、125.5 mg、126 mg、126.5 mg、127 mg、127.5 mg、128 mg、128.5mg、129 mg、129.5 mg、130 mg、130.5 mg、131 mg、131.5 mg、132 mg、132.5 mg、133 mg、133.5 mg、134 mg、134.5 mg、135 mg、135.5 mg、136 mg、136.5 mg、137 mg、137.5 mg、138mg、138.5 mg、139 mg、139.5 mg、140 mg、140.5 mg、141 mg、141.5 mg、142 mg、142.5 mg、143 mg、143.5 mg、144 mg、144.5 mg、145 mg、145.5 mg、146 mg、146.5 mg、147 mg、147.5mg、148 mg、148.5 mg、149 mg、149.5 mg、150 mg、150.5 mg、151 mg、151.5 mg、152 mg、152.5 mg、153 mg、153.5 mg、154 mg、154.5 mg、155 mg、155.5 mg、156 mg、156.5 mg、157mg、157.5 mg、158 mg、158.5 mg、159 mg、159.5 mg、160 mg、160.5 mg、161 mg、161.5 mg、162 mg、162.5 mg、163 mg、163.5 mg、164 mg、164.5 mg、165 mg、165.5 mg、166 mg、166.5mg、167 mg、167.5 mg、168 mg、168.5 mg、169 mg、169.5 mg、170 mg、170.5 mg、171 mg、171.5 mg、172 mg、172.5 mg、173 mg、173.5 mg、174 mg、174.5 mg、175 mg、175.5 mg、176mg、176.5 mg、177 mg、177.5 mg、178 mg、178.5 mg、179 mg、179.5 mg、180 mg、180.5 mg、181 mg、181.5 mg、182 mg、182.5 mg、183 mg、183.5 mg、184 mg、184.5 mg、185 mg、185.5mg、186 mg、186.5 mg、187 mg、187.5 mg、188 mg、188.5 mg、189 mg、189.5 mg、190 mg、190.5 mg、191 mg、191.5 mg、192 mg、192.5 mg、193 mg、193.5 mg、194 mg、194.5 mg、195mg、195.5 mg、196 mg、196.5 mg、197 mg、197.5 mg、198 mg、198.5 mg、199 mg、199.5 mg、200 mg、200.5 mg、201 mg、201.5 mg、202 mg、202.5 mg、203 mg、203.5 mg、204 mg、204.5mg、205 mg、205.5 mg、206 mg、206.5 mg、207 mg、207.5 mg、208 mg、208.5 mg、209 mg、209.5 mg、210 mg、210.5 mg、211 mg、211.5 mg、212 mg、212.5 mg、213 mg、213.5 mg、214mg、214.5 mg、215 mg、215.5 mg、216 mg、216.5 mg、217 mg、217.5 mg、218 mg、218.5 mg、219 mg、219.5 mg、220 mg、220.5 mg、221 mg、221.5 mg、222 mg、222.5 mg、223 mg、223.5mg、224 mg、224.5 mg、225 mg、225.5 mg、226 mg、226.5 mg、227 mg、227.5 mg、228 mg、228.5 mg、229 mg、229.5 mg、230 mg、230.5 mg、231 mg、231.5 mg、232 mg、232.5 mg、233mg、233.5 mg、234 mg、234.5 mg、235 mg、235.5 mg、236 mg、236.5 mg、237 mg、237.5 mg、238 mg、238.5 mg、239 mg、239.5 mg、240 mg、240.5 mg、241 mg、241.5 mg、242 mg、242.5mg、243 mg、243.5 mg、244 mg、244.5 mg、245 mg、245.5 mg、246 mg、246.5 mg、247 mg、247.5 mg、248 mg、248.5 mg、249 mg、249.5 mg、250 mg、250.5 mg、251 mg、251.5 mg、252mg、252.5 mg、253 mg、253.5 mg、254 mg、254.5 mg、255 mg、255.5 mg、256 mg、256.5 mg、257 mg、257.5 mg、258 mg、258.5 mg、259 mg、259.5 mg、260 mg、260.5 mg、261 mg、261.5mg、262 mg、262.5 mg、263 mg、263.5 mg、264 mg、264.5 mg、265 mg、265.5 mg、266 mg、266.5 mg、267 mg、267.5 mg、268 mg、268.5 mg、269 mg、269.5 mg、270 mg、270.5 mg、271mg、271.5 mg、272 mg、272.5 mg、273 mg、273.5 mg、274 mg、274.5 mg、275 mg、275.5 mg、276 mg、276.5 mg、277 mg、277.5 mg, 278 mg, 278.5 mg, 279 mg, 279.5 mg, 280 mg, 280.5 mg, 281 mg, 281.5 mg, 282 mg, 282.5 mg, 283 mg, 283.5 mg, 284 mg, 284.5 mg, 285 mg, 285.5 mg, 286 mg, 286.5 mg, 287 287.5 mg mg, 297 mg, 297.5 mg, 298 mg, 298.5 mg, 299 mg, 299.5mg or 300 mg. .

[0302] In various embodiments, the formulation comprises L-histidine, histidine monohydrochloride monohydrate, trehalose, mannitol, methionine, polysorbate 80, and optionally sterile water for injection (WFI). In various embodiments, the formulation comprises citric acid, sodium citrate, trehalose, mannitol, methionine, polysorbate 80, and optionally sterile water for injection (WFI).

[0303] This disclosure also provides kits containing, for example, bottles, vials, ampoules, test tubes, cartridges, and / or syringes, comprising liquid (e.g., sterile injectable) or solid (e.g., lyophilized) formulations. The kits may also contain pharmaceutically acceptable mediators or carriers (e.g., solvents, solutions, and / or buffers) for reconstituted solid (e.g., lyophilized) formulations into solutions or suspensions for administration (e.g., by injection), including but not limited to reconstituted lyophilized formulations in syringes or diluted concentrates to lower concentrations. Furthermore, ready-to-use injectable solutions and suspensions may be prepared from, for example, sterile powders, granules, or tablets comprising CNP-containing compositions. The kits may also include dispensing devices (e.g., aerosol or injection dispensing devices), pen syringes, auto-injectors, needle-free injectors, syringes, and / or needles.

[0304] As a non-limiting example, the kit may include a single-chamber or dual-chamber syringe. For a single-chamber syringe, the single chamber may contain a liquid CNP formulation ready for injection, or a solid (e.g., lyophilized) CNP formulation, or a CNP variant in a relatively small amount of a suitable solvent system (e.g., glycerol), which can be reconstituted into an injectable solution or suspension. For a dual-chamber syringe, one chamber may contain a pharmaceutically acceptable mediator or carrier (e.g., a solvent system, solution, or buffer), and the other chamber may contain a solid (e.g., lyophilized) CNP formulation, or a CNP variant in a relatively small amount of a suitable solvent system (e.g., glycerol), which can be reconstituted into an injectable solution or suspension using the mediator or carrier in the first chamber.

[0305] As another example, the kit may include one or more pen syringes or autoinjector devices, and a dual-chamber cartridge. One chamber of the cartridge may contain a pharmaceutically acceptable medium or carrier (e.g., a solvent system, solution, or buffer), and the other chamber may contain a solid (e.g., lyophilized) CNP formulation, or a CNP variant in a relatively small amount of a liquid formulation suitable for a solvent system (e.g., glycerol), which can be reconstituted using the medium or carrier in the first chamber into an injectable solution or suspension. The cartridge may contain an amount of the CNP variant sufficient to administer over a desired time period (e.g., 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, etc.). The pen syringe or autoinjector can be adjusted to administer the desired amount of the CNP formulation from the cartridge.

[0306] Application and administration

[0307] CNP variants or pharmaceutical compositions or formulations containing CNP variants may be administered to subjects in various ways, such as subcutaneously, intra-articularly, intraperitoneally, intramuscularly, intradermally, or orally. In one embodiment, the CNP variant composition is administered once daily, once weekly, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months.

[0308] CNP variants or combinations thereof can also be administered by implanting a drug reservoir into a target site of action, such as an abnormal or degenerated joint or cartilage region.

[0309] Alternatively, CNP variants can be administered sublingually (e.g., via a patch on the skin) or orally in the form of microspheres, microcapsules, liposomes (uncharged or charged (e.g., cationic)), polymer microparticles (e.g., polyamide, polylactide, polyglycolic acid, poly(lactide-glycolic acid), microemulsions, etc.

[0310] The CNP variant compositions described herein can be administered in therapeutically effective doses to patients in need to treat, improve, or prevent bone-related conditions (such as skeletal dysplasia, including achondroplasia). The safety and therapeutic efficacy of the CNP variants can be determined through standard pharmaceutical procedures in cell cultures or laboratory animals, for example, by determining the LD50. 50 (50% lethal dose for the population) and ED 50 (The dose that is effective for 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50. 50 / ED 50 Active agents exhibiting a large therapeutic index are generally preferred.

[0311] In some embodiments, the CNP variant compositions described herein are administered at doses ranging from about 3, 4, 5, 6, 7, 8, 9 or 10 nmol / kg to about 300 nmol / kg, or from about 20 nmol / kg to about 200 nmol / kg. In some embodiments, the CNP composition is administered at a dose of about 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750, or 2000 nmol / kg, or other doses deemed appropriate by the treating physician. In other embodiments, the CNP variant composition is prepared at about 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 pg / kg, or about 0.5, 0.8, 1.0, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 The CNP prodrug is administered at a dose of mg / kg, or at another dose deemed appropriate by the treating physician. In various embodiments, the CNP prodrug is administered at doses of about 5 μg / kg to 500 μg / kg, about 15 μg / kg to 350 μg / kg, about 25 μg / kg to 300 μg / kg, about 50 μg / kg to 250 μg / kg, or about 75 μg / kg to 200 μg / kg.In various embodiments, the CNP variants are administered at doses of approximately 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 35 μg / kg, 40 μg / kg, 45 μg / kg, 50 μg / kg, 60 μg / kg, 70 μg / kg, 75 μg / kg, 80 μg / kg, 90 μg / kg, 100 μg / kg, 125 μg / kg, 150 μg / kg, 175 μg / kg, 200 μg / kg, 225 μg / kg, 250 μg / kg, 275 μg / kg, 300 μg / kg, 325 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, or 500 μg / kg. The dosage of the CNP or CNP variant described herein may be administered according to the dosing / application frequency described herein, including but not limited to once daily, twice or three times weekly, once weekly, once every two weeks, once every three weeks, once monthly, etc. In various embodiments, the CNP or CNP variant is administered subcutaneously daily. In various embodiments, the CNP or CNP variant is administered subcutaneously weekly. In various embodiments, the CNP variant is administered at a dose of 2.5 pg / kg to 60 pg / kg daily, 10 pg / kg to 45 pg / kg daily, or 15 pg / kg to 30 pg / kg daily. In various embodiments, the CNP variant is administered at a dose of 15 pg / kg daily. In various embodiments, the CNP variant is administered at a dose of 30 pg / kg daily.

[0312] In various embodiments, the pharmaceutical composition is a sustained-release composition. In various embodiments, compared with non-sustained-release compositions comprising the same active CNP compound (i.e., free drug), pharmaceutical compositions comprising a CNP prodrug have a longer half-life, improved Cmax, and improved AUC.

[0313] The frequency of administration / application of CNP variants for a specific subject can vary depending on a number of factors, including the condition being treated and the subject's condition and response to the therapy. CNP variants can be administered as a single dose or multiple doses per administration. In some embodiments, the CNP variant composition is administered as a single dose or multiple doses, once daily, once weekly, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months, or as deemed appropriate by the treating physician. In various embodiments, the CNP variant is administered for 3 months, 6 months, 12 months, or longer.

[0314] In some embodiments, a CNP variant composition is administered to allow a growth phase (e.g., cartilage formation), followed by a recovery phase (e.g., osteogenic formation). For example, the CNP composition may be administered subcutaneously or via another modality once daily or multiple times weekly for a period of time, followed by a treatment-free period, which is then repeated. In some embodiments, the initial treatment period (e.g., administration of the CNP variant composition once daily or multiple times weekly) lasts for 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In one related embodiment, the treatment-free period lasts for 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the CNP variant composition is administered once daily for 3 days, followed by a 3-day break; or once daily or multiple times weekly for 1 week, followed by a 3-day or 1-week break; or once daily or multiple times weekly for 2 weeks, followed by a 1- or 2-week break; or once daily or multiple times weekly for 3 weeks, followed by a 1, 2, or 3-week break; or once daily or multiple times weekly for 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, followed by a 1, 2, 3, or 4-week break.

[0315] biomarkers

[0316] To treat bone-related conditions, growth indicators can be measured, such as long bone growth measurements in utero and in newborns; and bone growth biomarkers can be measured, such as CNP, cGMP, collagen II, collagen X, osteocalcitonin, and proliferating cell nuclear antigen (PCNA).

[0317] One marker of CNP signaling is cGMP (3',5'-cyclic guanosine monophosphate). The level of this intracellular signaling molecule increases after binding to CNP and activation of its homologous receptor, NPR-B. Elevated levels of cGMP can be measured in cell culture extracts following CNP exposure (in vitro), in conditioned media from bone explant studies following CNP exposure (ex vivo), and in plasma within minutes of CNP administration via subcutaneous, intravenous, or other routes of administration known in the art (in vivo).

[0318] Cartilage and bone-specific analytes (or cartilage and bone-related markers) can also be measured to assess CNP efficacy. For example, fragments of cleaved type II collagen are cartilage-specific markers related to cartilage turnover. Type II collagen is the main organic component of cartilage, and fragments of type II collagen (cleaved collagen) are released into circulation and subsequently secreted into the urine after cartilage metabolism. Cartilage metabolism precedes new bone formation.

[0319] Measurable bone-specific biomarkers related to bone formation include the N-terminal propeptide (PINP) of type I collagenogen. Type I collagen synthesis is a crucial step in bone formation, as it is a major organic component of the bone matrix. During collagen synthesis, the propeptide is released from the collagenogen molecule and can be detected in serum. Additionally, fragments of type I collagen can be measured as markers related to bone resorption.

[0320] Other potential biomarkers related to cartilage and bone formation and growth include chondroitin sulfate (a cartilage-specific marker related to cartilage metabolism), propeptide of type II collagen (a cartilage-specific marker related to cartilage formation), C-terminal peptide of type I collagen (CTx), alkaline phosphatase (bone-specific), and osteocalcitonin (a bone-specific marker related to bone formation). Biomarkers also include proliferating cell nuclear antigen (PCNA), propeptide and fragments of type I collagen, type I collagen and fragments of collagen, chondroitin sulfate, collagen X, CXM (non-collagen 1 (NC1) domain of type X collagen), NTproCNP and alkaline phosphatase, N-terminal type I collagen propeptide, bone-specific alkaline phosphatase, N-terminal propeptide of type I collagen / N-propeptide of type I collagen (PINP), cross-linked type I collagen C-terminal peptide (CTx), cross-linked type I collagen N-terminal peptide (NTx) tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomic readouts (e.g., from PAXgene® RNA), and CNP-variant bioactivity. Cartilage-related and bone-related biomarkers can be measured using commercially available kits, for example, in serum from conditioned media from efficacy / pharmacodynamic studies and from in vitro studies.

[0321] In one embodiment, the level of at least one bone-related or cartilage-related biomarker is determined or measured in subjects who have been administered the CNP variant or composition described herein in order to monitor the effect of the CNP composition on bone and cartilage formation and growth in vivo. For example, an increase in the level of at least one bone-related or cartilage-related biomarker may indicate a positive effect of administration of the CNP variant or composition on bone growth and may be used to treat skeletal dysplasia and other bone-related or cartilage-related diseases or conditions associated with reduced CNP activity.

[0322] Exemplary bone-related or cartilage-related biomarkers include, but are not limited to, CNP (e.g., endogenous CNP levels), cGMP, type II collagen propeptide and fragments, type II collagen and fragments, type I collagen C-terminal peptide (CTx), osteocalcitonin, proliferating cell nuclear antigen (PCNA), type I collagen precursor propeptide (PINP) and fragments, type I collagen and fragments, agglutinin-glucan chondroitin sulfate, and alkaline phosphatase.

[0323] In various embodiments, the at least one bone-related or cartilage-related biomarker is selected from the group consisting of: CNP, cGMP, type II collagen propeptide and fragment thereof, type II collagen and fragment thereof, type I collagen C-terminal peptide (CTx), osteocalcitonin, proliferating cell nuclear antigen (PCNA), type I collagen propeptide and fragment thereof, type I collagen and fragment thereof, agglutinin sulfate chondroitin, collagen X, CXM (non-collagen 1 (NC1) domain of type X collagen), NTproCNP and alkaline phosphatase, N-terminal type I collagen propeptide, bone-specific alkaline phosphatase, type I collagen N-terminal propeptide / type I collagen propeptide N-propeptide (PINP), cross-linked type I collagen C-terminal peptide (CTx), cross-linked type I collagen N-terminal peptide (NTx) tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomic readouts (e.g. from PAXgene® RNA), and CNP-variant bioactivity. NTproCNP is the N-terminal propeptide of CNP (NTproCNP), which is released from cells in an equimolar ratio with CNP. The biologically active form of CNP appears in low concentrations in plasma due to the rapid clearance rate of the peptide. NTproCNP is not cleared via the same mechanism and has been found in circulation at concentrations 20 to 50 times higher (Olney et al., Clin Endocrinol (Oxf.) 2012, 77:416-422).

[0324] Type X collagen biomarkers (CXMs) are degradation fragments of type X collagen that contain the intact trimeric non-collagenous 1 (NC1) domain of type X collagen. CXMs are released by the active growth plate and decrease in the sample with age. CXM levels are correlated with growth rate in children (Coghlan et al., Sci Translational Medicine 2017, 9(419):eaan4669).

[0325] Bone-specific alkaline phosphatase (BSAP or BAP) is a bone growth biomarker produced by osteoblasts and osteoclasts in the growth plate and mineralized bone. Changes in BSAP can reflect growth plate activity, bone growth, and / or bone remodeling.

[0326] Measurable bone-specific biomarkers related to bone formation include the N-terminal propeptide of type I collagenogen (PINP). Type I collagen synthesis is a crucial step in bone formation, as it is a major organic component of the bone matrix. During collagen synthesis, the propeptide is released from the collagenogen molecule and can be detected in serum. Additionally, fragments of type I collagen can be measured as markers related to bone resorption.

[0327] Other potential biomarkers related to cartilage and bone formation and growth include chondroitin sulfate (a cartilage-specific marker related to cartilage metabolism), propeptide of type II collagen (a cartilage-specific marker related to cartilage formation), C-terminal peptide (CTx) of type I collagen, alkaline phosphatase (bone-specific), and osteocalcitonin (a bone-specific marker related to bone formation). Cartilage-related and bone-related biomarkers can be measured using commercially available kits, for example, in serum from conditioned media obtained from efficacy / pharmacodynamic studies in vivo and from in vitro studies.

[0328] In various embodiments, biomarkers are measured by obtaining biological samples from subjects who will be, are being, or have been given a CNP variant. Biomarkers can be measured using techniques known in the art, including but not limited to Western blotting, enzyme-linked immunosorbent assay (ELISA), and enzyme activity assays. Biological samples can be blood, serum, urine, or other biological fluids.

[0329] preparation

[0330] In other embodiments, this disclosure covers the use of pharmaceutical compositions and formulations comprising CNP variant peptides and one or more pharmaceutically acceptable excipients, carriers, and / or diluents. In some embodiments, the compositions also comprise one or more other bioactive agents (e.g., proteases, receptor tyrosine kinases, and / or inhibitors that clear receptor NPR-C).

[0331] Some possible formulations are disclosed in the Examples section. In any formulation, the concentration of the CNP variant (in mg / mL) is typically 0.5 or higher, 1 or higher, 5 or higher, 10 or higher, 15 or higher, 20 or higher, 25 or higher, or even 30 or higher. In any formulation, the concentration of the CNP variant (in mg / mL) is typically 40 or lower, 35 or lower, 30 or lower, 25 or lower, 20 or lower, 15 or lower, 10 or lower, or even 5 or lower.

[0332] Non-limiting examples of excipients, carriers, and diluents include mediators, liquids, buffers, isotonic agents, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, adjuvants, etc. The composition may contain liquids (e.g., water, ethanol); diluents having various buffer contents (e.g., Tris-HCl, phosphate, acetate buffers, citrate buffers), pH, and ionic strength; detergents and solubilizers (e.g., polysorbate 20, polysorbate 80 (sometimes referred to as PS 80)); antioxidants (e.g., methionine, ascorbic acid, sodium metabisulfite); preservatives (e.g., thimerosal, benzyl alcohol, m-cresol); and additives (e.g., lactose, mannitol, sucrose). The use of excipients, diluents and carriers in the formulation of pharmaceutical compositions is known in the art; see, for example, Remington Pharmaceutical Science, 18th edition, pp. 1435-1712, Mack Publishing Co. (Easton, Pennsylvania (1990)), which is incorporated herein by reference in its entirety.

[0333] For example, carriers include, but are not limited to, diluents, mediators, and adjuvants, as well as implant carriers, and inert, non-toxic solid or liquid fillers and encapsulating materials that do not react with one or more active ingredients. Non-limiting examples of carriers include phosphate-buffered saline, physiological saline, water, and emulsions (e.g., oil / water emulsions). Carriers can be solvents or dispersion media containing, for example, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, and mixtures thereof.

[0334] In some embodiments, the composition is a liquid formulation. In some embodiments, the formulation comprises a CNP variant peptide at a concentration in the following ranges: about 0.1 mg / ml to about 20 mg / ml, or about 0.5 mg / ml to about 20 mg / ml, or about 1 mg / ml to about 20 mg / ml, or about 0.1 mg / ml to about 10 mg / ml, or about 0.5 mg / ml to about 10 mg / ml, or about 1 to 10 mg / ml, or about 2 mg / ml to about 10 mg / ml. In other embodiments, the formulation may be a lyophilized formulation or a liquid formulation pre-reconstituted from a lyophilized formulation.

[0335] In other embodiments, the composition comprises a buffer solution or buffering agent to maintain the pH of the CNP-containing solution or suspension within a desired range. Non-limiting examples of buffer solutions include phosphate-buffered saline, Tris-buffered saline, and Hankley-buffered saline. Buffering agents include, but are not limited to, sodium acetate, sodium phosphate, citrate monohydrate, and sodium citrate dihydrate. Mixtures of buffering agents may also be used. In some embodiments, the buffering agent is acetate / acetate or citric acid / citrate. The suitable amount of buffering agent in the composition depends in part on the specific buffering agent used and the desired pH of the solution or suspension. For example, acetate is a more effective pH buffering agent at pH 5 than at pH 6, so less acetate may be used in a solution at pH 5 than in a solution at pH 6. In some embodiments, the concentration of the buffering agent is about 5-15 mM (e.g., 10 mM + 5 mM). In some embodiments, the pH of the composition is from about pH 3 to about pH 7.5, or from about pH 3.5 to about pH 7, or from about pH 3.5 to about pH 6.5, or from about pH 4 to about pH 6, or from about pH 4 to about pH 5, or about pH 5.0 ± 1.0, or about pH 5.5 + 1.0.

[0336] In other embodiments, the composition contains an isotonic adjuster to make the solution or suspension isotonic and more suitable for injection. Non-limiting examples of isotonic agents include NaCl, trehalose, mannitol, dextrose, glucose, glycerol, sorbitol, xylitol, and ethanol. In some embodiments, the isotonic agent is trehalose or mannitol, which may be used alone or in combination. In some embodiments, the concentration of trehalose or mannitol is about 160 ± 20 mM, or about 140 mM ± 20 mM, or about 120 ± 20 mM, or about 100 mM ± 20 mM, or about 80 mM ± 20 mM, or about 60 mM ± 20 mM. The ratio of trehalose to mannitol may be about 4:1, for example about 3:1 to about 5:1.

[0337] In various embodiments, the composition may contain a preservative. Preservatives include, but are not limited to, m-cresol and benzyl alcohol. In some embodiments, the concentration of the preservative is about 0.4% + 0.2%, or about 1% + 0.5%, or about 1.5% + 0.5%, or about 2.0% + 0.5%. In some embodiments of the invention, the composition or formulation does not contain a preservative.

[0338] In various embodiments, the composition contains an anti-adsorption agent (e.g., to reduce the adsorption of CNP variants onto glass or plastic). Anti-adsorption agents include, but are not limited to, benzyl alcohol, polysorbate 20, and polysorbate 80. In some embodiments, the concentration of the anti-adsorption agent is about 0.001% to about 0.5%, or about 0.01% to about 0.5%, or about 0.1% to about 1%, or about 0.5% to about 1%, or about 0.5% to about 1.5%, or about 0.5% to about 2%, or about 1% to about 2%.

[0339] In various embodiments, the composition comprises a stabilizer. Non-limiting examples of stabilizers include glycerol, glycerol, thioglycerol, methionine, and ascorbic acid and its salts. In some embodiments, when the stabilizer is thioglycerol or ascorbic acid or its salts, the concentration of the stabilizer is from about 0.1% to about 1%.

[0340] In various embodiments, the composition contains an antioxidant. Exemplary antioxidants are not limited to ascorbic acid. In some embodiments, the molar ratio of the antioxidant to the CNP variant peptide is about 0.1:1 to about 15:1, or about 1:1 to about 15:1, or about 0.5:1 to about 10:1, or about 1:1 to about 10:1, or about 3:1 to about 10:1.

[0341] Pharmaceutically acceptable salts may be used in the compositions, including but not limited to inorganic acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate), organic acid salts (e.g., acetate, propionate, malonate, benzoate, methanesulfonate, toluenesulfonate), and amine salts (e.g., isopropylamine, trimethylamine, dicyclohexylamine, diethanolamine). A detailed discussion of pharmaceutically acceptable salts can be found in Remington Pharmaceutical Science, 18th edition, Mack Publishing Company (Easton, Pennsylvania (1990)).

[0342] Formulations for parenteral administration can be prepared, for example, in the form of liquid solutions or suspensions, solid forms suitable for dissolving or suspending in a liquid medium prior to injection, or emulsions. For example, sterile injectable solutions and suspensions can be formulated using suitable diluents, carriers, solvents (e.g., buffered aqueous solutions, Ringer's solutions, isotonic sodium chloride solutions), dispersants, wetting agents, emulsifiers, suspending agents, etc., according to techniques known in the art. Additionally, sterile non-volatile oils, fatty esters, polyols, and / or other inactive ingredients may be used. As another example, formulations for parenteral administration include aqueous sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners.

[0343] Exemplary formulations containing CNP peptides are described in U.S. Patents 8,198,242 and 8,598,121. These cover formulations using CNP peptides with a pH ranging from about 4 to about 6.

[0344] In various embodiments, the CNP variant peptide can be formulated in a pharmaceutically acceptable carrier for administration to subjects affected by skeletal dysplasia. In some embodiments, the liquid formulation of the CNP variant peptide is formulated according to any combination of the ingredients, and the amounts or concentrations thereof are described below.

[0345] Compositions containing CNP variant peptides may also be lyophilized formulations. In some embodiments, the lyophilized formulations contain buffers and build-up agents, and optionally antioxidants. Exemplary buffers include, but are not limited to, acetate buffers and citrate buffers. Exemplary build-up agents include, but are not limited to, mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVPK24). In some embodiments, the amount of mannitol and / or trehalose is about 3% to about 10%, or about 4% to about 8%, or about 4% to about 6%. In some embodiments, the amount of sucrose is about 6% to about 20%, or about 6% to about 15%, or about 8% to about 12%.

[0346] In various embodiments, the lyophilized formulations of CNP variant peptides are prepared from formulations formulated according to any combination of the ingredients and their amounts or concentrations described below.

[0347] In various embodiments, the formulations containing the CNP variant peptide have a pH of about 3-7, or about 3-6, or about 3.5-6.5, or about 4-6, or about 4-5, or about 4.5-5.5. In some embodiments, for pH 4-5.5, a suitable buffer is acetate / acetate (e.g., sodium acetate), and for pH 5.5-6, a suitable buffer is citric acid / citrate. Citric acid / citrate (e.g., sodium citrate) is also a suitable buffer in the pH range of 3-6 or 4-6. In some embodiments, the concentration of the buffer in the formulation is about 2-50 mM, or about 2-40 mM, or about 2-30 mM, or about 5-30 mM, or about 2-20 mM, or about 5-20 mM, or about 5-15 mM.

[0348] Additionally, to minimize or avoid deamidation of the CNP variant peptide, water in the formulation can be removed by lyophilization. In some embodiments, the lyophilized formulation contains any combination of the following components: buffer: sodium acetate and acetic acid, or sodium citrate and citric acid; isotonic agent / accumulator: mannitol (e.g., 3-10%, 2-8%, or 4-6%); sucrose (e.g., 6-20%, 5-15%, or 8-12%); antioxidant: methionine and / or ascorbic acid, wherein the molar ratio of each antioxidant to the CNP variant peptide is about 0.1:1 to about 1:1, or about 0.5:1 to about 5:1, or about 1:1 to about 15:1, or about 1:1 to about 10:1, or about 3:1 to about 10:1.

[0349] Deamidation can also be minimized or avoided by storing CNP compositions (e.g., liquid or lyophilized formulations) at lower temperatures, such as about 5°C, 0°C, -10°C, -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, or -100°C.

[0350] To minimize or avoid oxidation of oxidizable residues (e.g., methionine) in CNP variant peptides, the variants can be formulated with one or more antioxidants. Exemplary antioxidants include, but are not limited to, methionine, ascorbic acid, and thioglycerol. Oxidation of residues, such as methionine, can also be minimized or prevented by purging oxygen from the liquid medium (if it is a liquid formulation) with nitrogen or argon, and / or by purging oxygen from the container or packaging with nitrogen or argon.

[0351] In some embodiments, to minimize or prevent adsorption (e.g., adsorption of CNP variant peptides onto plastics or glass), polysorbate 20, polysorbate 80, or benzyl alcohol, or combinations thereof, are added to the CNP formulation. In some embodiments, the concentration of each of one or more anti-adsorbents is about 0.001% to about 0.5%, or about 0.01% to about 0.5%, or about 0.1% to about 1%, or about 0.5% to about 1%, or about 0.5% to about 1.5%, or about 0.5% to about 2%, or about 1% to about 2%. Exemplary ranges of anti-adsorbents in the formulation include, but are not limited to, about 0.001% to about 0.5% of polysorbate 20, about 0.001% to about 0.5% of polysorbate 80, and / or about 0.5% to about 1.5% of benzyl alcohol.

[0352] This disclosure also provides kits containing, for example, bottles, vials, ampoules, test tubes, cartridges, and / or syringes, comprising liquid (e.g., sterile injectable) or solid (e.g., lyophilized) formulations. The kits may also contain pharmaceutically acceptable mediators or carriers (e.g., solvents, solutions, and / or buffers) for reconstituted solid (e.g., lyophilized) formulations into solutions or suspensions for administration (e.g., by injection), including but not limited to reconstituted lyophilized formulations in syringes or diluting concentrates to lower concentrations. Furthermore, ready-to-use injectable solutions and suspensions can be prepared from, for example, sterile powders, granules, or tablets comprising CNP-containing compositions. The kits may also include dispensing devices (e.g., aerosol or injection dispensing devices), pen syringes, auto-injectors, needle-free injectors, syringes, and / or needles.

[0353] Other aspects and details of this disclosure will become apparent from the following examples, which are intended to be illustrative and not restrictive.

[0354] Example

[0355] 1. A pharmaceutical composition comprising a variant of C-type natriuretic peptide (CNP) PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:1), a pharmaceutically acceptable excipient, and a carrier or diluent, wherein the CNP variant comprises an acid moiety, a spacer, a hydrolyzable linker, and is delivered via... Figure 11 The structural characterization shown is illustrated.

[0356] 2. A pharmaceutical composition comprising (4R, 10S, 16S, 19S, 22S, 28S, 31S, 34S, 37S, 40S, 43S, 49S, 52R)-52-(2-((S)- ... -2-((S)-1-(L-prolyl-glycyl-L-glutamine-l-glutamine-L-histyl-pyrrolidine-2-carboxamido)-4-amino-4-oxobutamido)propamido)-5-guanidinylpentamido)-6-aminohexanoyl)-3-(4-hydroxyphenyl)propamido)-6-aminohexanoyl)acetamyl)propamido)-4-amino-4-oxobutamido)-6-aminohexanoyl)acetamyl)-6-aminohexanoyl)acetamyl)- 4-Methylpentamido)-3-hydroxypropamido)-6-aminohexano)acetami)-49-benzyl-28-((S)-sec-butyl)-34-(carboxymethyl)-40-((S)-33,51-dicarboxy-8-(2-hydroxyethyl)-6,12,21,30,35-pentoxo-14,17,23,26-tetraoxa-5,8,11,20,29,34-hexaazapentaylyl)-31-(3-guanidinopropyl)-16,22-bis( Hydroxymethyl)-10,37,43-triisobutyl-19-(2-(methylthio)ethyl)-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-hexadecoxo-1,2-dithia-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-hexaazacyclopentatriane-4-carboxylic acid, pharmaceutically acceptable excipients and carriers or diluents.

[0357] 3. A pharmaceutical composition as described in Example 1 or 2, wherein the pharmaceutical composition is lyophilized.

[0358] 4. A pharmaceutical composition as described in any one of Examples 1 to 3, wherein the carrier or diluent comprises a buffer.

[0359] 5. The pharmaceutical composition of Example 4, wherein the buffer comprises a buffer selected from the group consisting of citrate, acetate, phosphate, TRIS, and combinations thereof.

[0360] 6. The pharmaceutical composition of Example 5, wherein the buffer further comprises histidine, its salt, its solvate, or a solvate of a salt thereof.

[0361] 7. The pharmaceutical composition of any one of Examples 4 to 6, wherein the buffer is present at a concentration of 5-15 mM.

[0362] 8. The pharmaceutical composition of any one of Examples 4 to 7 has a pH of 3-9.

[0363] 9. The pharmaceutical composition of Example 8 has a pH of 4-6.

[0364] 10. The pharmaceutical composition of Example 9 has a pH of 5-6.

[0365] 11. The pharmaceutical composition of Example 10 has a pH of 5.2 or 5.5.

[0366] 12. A pharmaceutical composition as described in any one of Examples 1 to 11, wherein the pharmaceutically acceptable excipient is selected from bulking agents, tension modifiers, antioxidants, surfactants, solubilizers, stabilizers, and combinations thereof.

[0367] 13. The pharmaceutical composition of Example 12, wherein the accumulator is selected from mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24), and combinations thereof.

[0368] 14. A pharmaceutical composition as described in Example 12 or 13, wherein the accumulator comprises trehalose or a solvate thereof, mannitol or a combination thereof.

[0369] 15. The pharmaceutical composition of Example 14, wherein the accumulator comprises trehalose and mannitol in a weight ratio of 3:1 to 1:1.

[0370] 16. The pharmaceutical composition of Example 15, wherein the weight ratio of trehalose to mannitol is 3.9:1.

[0371] 17. A pharmaceutical composition as described in any one of Examples 13 to 16, wherein trehalose is present in an amount of 3-6% by weight of the composition.

[0372] 18. The pharmaceutical composition of Example 17, wherein trehalose is present in an amount of 3.5-5.8% by weight of the composition.

[0373] 19. The pharmaceutical composition of Example 18, wherein trehalose is present in an amount of 3.8-4.8% by weight of the composition.

[0374] 20. A pharmaceutical composition of any one of Examples 12 to 19, wherein the pharmaceutical composition comprises a tension modifier selected from sodium chloride, dextran, glucose, glycerol, sorbitol, xylitol, ethanol, and combinations thereof.

[0375] 21. A pharmaceutical composition of any one of Examples 12 to 20, wherein the pharmaceutical composition comprises an antioxidant selected from methionine, ascorbic acid, salts of ascorbic acid, thioglycerol, and combinations thereof.

[0376] 22. The pharmaceutical composition of Example 21, wherein the antioxidant is methionine.

[0377] 23. A pharmaceutical composition as described in any of Examples 1 to 22, comprising a stabilizer or surfactant selected from glycine, sorbitol, polysorbate, and combinations thereof.

[0378] 24. The pharmaceutical composition of Example 23, wherein the pharmaceutical composition comprises polysorbate.

[0379] 25. A pharmaceutical composition of any one of Examples 1 to 24, wherein the pharmaceutical composition comprises L-histidine, histidine monohydrochloride monohydrate, trehalose dihydrate, D-mannitol, L-methionine, and polysorbate.

[0380] 26. A pharmaceutical composition of any one of Examples 1 to 24, wherein the pharmaceutical composition comprises a citrate buffer, trehalose dihydrate, D-mannitol, L-methionine, and polysorbate.

[0381] 27. A pharmaceutical composition comprising (4R, 10S, 16S, 19S, 22S, 28S, 31S, 34S, 37S, 40S, 43S, 49S, 52R)-52-(2-((S)- ... -((S)-1-(L-prolyl-glycyl-L-glutamine-l-glutamine-L-histyl-pyrrolidine-2-carboxamido)-4-amino-4-oxobutamido)propamido)-5-guanidinylpentamido)-6-aminohexanoyl)-3-(4-hydroxyphenyl)propamido)-6-aminohexanoyl)acetamyl)propamido)-4-amino-4-oxobutamido)-6-aminohexanoyl)acetamyl)-4-methylpentamido) (amido)-3-hydroxypropamido)-6-aminohexamido)acetami)-49-benzyl-28-((S)-sec-butyl)-34-(carboxymethyl)-40-((S)-33,51-dicarboxy-8-(2-hydroxyethyl)-6,12,21,30,35-pentoxo-14,17,23,26-tetraoxa-5,8,11,20,29,34-hexaazapentadecyl)-31-(3-guanidinopropyl)-16,22-bis(hydroxymethyl)-10, 37,43-Triisobutyl-19-(2-(methylthio)ethyl)-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-Hexadecoxo-1,2-dithia-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-Hexaazacyclopentatriane-4-carboxylic acid, buffers containing histidine or its salts, and one or more pharmaceutically acceptable excipients.

[0382] 28. The pharmaceutical composition of Example 27, wherein the buffer comprises L-histidine hydrochloride monohydrate.

[0383] 29. The pharmaceutical composition of Example 27 or 28, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient selected from the group consisting of: build-up agents, stabilizers, anti-adsorption agents, diluents, and combinations thereof.

[0384] 30. A pharmaceutical composition of any one of Examples 27 to 29, wherein the pharmaceutically acceptable excipient comprises a bulking agent, the bulking agent comprising trehalose or a solvation thereof.

[0385] 31. The pharmaceutical composition of Example 30, wherein the accumulator comprises trehalose dihydrate.

[0386] 32. A pharmaceutical composition of any one of Examples 27 to 31, wherein a pharmaceutically acceptable excipient comprises a build-up agent, said build-up agent comprising mannitol.

[0387] 33. The pharmaceutical composition of Example 32, wherein the mannitol is D-mannitol.

[0388] 34. A pharmaceutical composition of any one of Examples 27 to 33, wherein the pharmaceutically acceptable excipient comprises a stabilizer comprising methionine.

[0389] 35. The pharmaceutical composition of Example 34, wherein the methionine is L-methionine.

[0390] 36. A pharmaceutical composition of any one of Examples 27 to 35, wherein the pharmaceutically acceptable excipient comprises an anti-adsorption agent, wherein the anti-adsorption agent comprises polysorbate.

[0391] 37. The pharmaceutical composition of Example 36, wherein the polysorbate is polysorbate 80.

[0392] 38. A pharmaceutical composition of any one of Examples 27 to 37, wherein the pharmaceutical composition is substantially free of citrate buffer.

[0393] 39. A pharmaceutical composition comprising a C-type natriuretic peptide (CNP) variant, namely (4R, 10S, 16S, 19S, 22S, 28S, 31S, 34S, 37S, 40S, 43S, 49S, 52R)-52-(2-((S)-2-((S)-2-((S)-2-(2-((S)-2-((S)-2-((S)-2-((S)-2-(2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-1-(L-prolylglycyl-L-glutamineyl-L-glutamineyl-L-histyl)pyrrolidine-2-carboxamido)-4-amino- 4-O-Butyramido)propamido)-5-Guidinopentamido)-6-Aminohexamido)-3-(4-Hydroxyphenyl)propamido)-6-Aminohexamido)acetamamido)propamido)-4-Amino-4-O-Butyramido)-6-Aminohexamido)-6-Aminohexamido)acetamamido)-4-Methylpentamido)-3-Hydroxypropamido)-6-Aminohexamido)acetamamido)-4,9-Benzyl-28-((S) -sec-butyl)-34-(carboxymethyl)-40-((S)-33,51-dicarboxy-8-(2-hydroxyethyl)-6,12,21,30,35-pentoxo-14,17,23,26-tetraoxa-5,8,11,20,29,34-hexaazapentadecyl)-31-(3-guanidinopropyl)-16,22-bis(hydroxymethyl)-10,37,43-triisobutyl-19-(2-(methylthio)ethyl)-6, 9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-hexadecoxo-1,2-dithia-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-hexaazacyclopentatriane-4-carboxylic acid; histidine buffer; trehalose dihydrate; D-mannitol; L-methionine; polysorbate 80; and water.

[0394] 40. The pharmaceutical composition of Example 39, wherein the CNP variant is present at a concentration of 10 mg / mL.

[0395] 41. The pharmaceutical composition of Example 39, wherein the CNP variant is present at a concentration of 30 mg / mL.

[0396] 42. The pharmaceutical composition of any one of Examples 39 to 41, wherein the histidine buffer comprises L-histidine and histidine monohydrochloride monohydrate.

[0397] 43. The pharmaceutical composition of Example 42, wherein L-histidine is present at a concentration of 0.35 mg / mL.

[0398] 44. The pharmaceutical composition of Example 42, wherein L-histidine is present at a concentration of 2.2 mM.

[0399] 45. The pharmaceutical composition of any one of Examples 42 to 44, wherein L-histidine monohydrochloride monohydrate is present at a concentration of 1.6 mg / mL.

[0400] 46. ​​The pharmaceutical composition of any one of Examples 42 to 44, wherein L-histidine monohydrochloride monohydrate is present at a concentration of 7.8 mM.

[0401] 47. The pharmaceutical composition of any one of Examples 39 to 46, wherein the trehalose dihydrate is present at a concentration of 58 mg / mL.

[0402] 48. The pharmaceutical composition of any one of Examples 39 to 46, wherein the trehalose dihydrate is present at a concentration of about 127 mM to about 153 mM.

[0403] 49. The pharmaceutical composition of Example 48, wherein the trehalose dihydrate is present at a concentration of 127 mM.

[0404] 50. The pharmaceutical composition of Example 48, wherein the trehalose dihydrate is present at a concentration of 153.3 mM.

[0405] 51. The pharmaceutical composition of any one of Examples 39 to 50, wherein the D-mannitol is present at a concentration of 15 mg / mL.

[0406] 52. The pharmaceutical composition of any one of Examples 39 to 50, wherein the D-mannitol is present at a concentration of about 68 mM to about 82 mM.

[0407] 53. The pharmaceutical composition of Example 52, wherein the D-mannitol is present at a concentration of 68.1 mM.

[0408] 54. The pharmaceutical composition of Example 52, wherein the D-mannitol is present at a concentration of 82.3 mM.

[0409] 55. The pharmaceutical composition of any one of Examples 39 to 54, wherein the L-methionine is present at a concentration of 0.7 mg / mL.

[0410] 56. The pharmaceutical composition of any one of Examples 39 to 54, wherein the L-methionine is present at a concentration of 4.9 mM.

[0411] 57. The pharmaceutical composition of any one of Examples 39 to 56, wherein the polysorbate 80 is present at a concentration of 0.05 mg / mL.

[0412] 58. The pharmaceutical composition of any one of Examples 39 to 56, wherein the polysorbate 80 is present at a concentration of 0.005% (v / v).

[0413] 59. A pharmaceutical composition as described in any one of Examples 1 to 58, wherein the composition exhibits a lower Cmax and a higher AUC compared to an equivalent composition lacking an acid moiety, spacer, and hydrolyzable linker.

[0414] 60. A pharmaceutical kit comprising a pharmaceutical composition as described in any one of Examples 1 to 58.

[0415] 61. A pharmaceutical kit comprising a pharmaceutical composition as described in any one of Examples 39 to 58, wherein the CNP variant is present in an amount of 13 mg.

[0416] 62. A pharmaceutical kit comprising a pharmaceutical composition as described in any one of Examples 39 to 58, wherein the CNP variant is present in an amount of 39 mg.

[0417] 63. A method for treating a subject with bone-related conditions or skeletal dysplasia, the method comprising administering to the subject a composition as described in any one of Examples 1 to 59.

[0418] 64. The method of Example 63, wherein the bone-related condition or skeletal dysplasia is selected from the group consisting of: osteoarthritis, hypophosphatemic rickets, achondroplasia, decreased cartilage production, short stature, dwarfism, osteochondrodysplasia, lethal achondroplasia, osteogenesis imperfecta, chondrodysplasia punctate chondrodysplasia, homozygous achondroplasia, brachydactyly, congenital lethal hypophosphatase syndrome, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, pedicled punctate chondrodysplasia, Janssen type metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, skeletal dysplasia, malformation dysplasia, congenital Short femur, Langer type mid-limb dysplasia, Nivig type mid-limb dysplasia, Robinnoc syndrome, Reinhardt syndrome, acrodysplasia, peripheral bone dysplasia, Knifell's dysplasia, fibrocartilage hyperplasia, Roberts syndrome, acromegaly, microlimb syndrome, Moquer syndrome, Knifell's syndrome, metaphyseal dysplasia and vertebral epiphyseal dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Lerreville's disease), PTPN11 mutation (Noonan syndrome), insulin growth factor 1 receptor (IGF1R) mutation, idiopathic short stature, and osteoporosis.

[0419] 65. A method for elongating bones or increasing long bone growth in a subject in need, the method comprising administering to the subject a composition as described in any one of Examples 1 to 59, wherein the administration causes bone elongation or increases long bone growth.

[0420] 66. The method of any one of Examples 63 to 64, wherein the composition is administered subcutaneously, intradermally, intra-articularly, orally, or intramuscularly.

[0421] 67. The method of any one of Examples 63 to 66, wherein the composition provides a prolonged release composition.

[0422] 68. The method of any one of Examples 63 to 66, wherein the composition is applied once every 5 days, once a week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months.

[0423] 69. A method for treating CNP-responsive conditions or symptoms, the method comprising:

[0424] -Administer the composition according to any one of Examples 1 to 58 to the subject, and

[0425] - Monitor the levels of at least one bone-related or cartilage-related biomarker in the subjects.

[0426] An increase in the level of at least one bone-related or cartilage-related biomarker indicates that the CNP variant has a therapeutic effect on the subject or the condition or symptom.

[0427] 70. The method of Example 69, further comprising adjusting the amount or frequency of application of the composition, wherein...

[0428] i) If the level of at least one bone-related or cartilage-related biomarker is below the target level, increase the dosage or frequency of the composition; or

[0429] ii) If the level of at least one bone-related or cartilage-related biomarker is higher than the target level, reduce the amount or frequency of application of the composition.

[0430] 71. The method of Example 69 or 70, wherein the at least one bone-related or cartilage-related biomarker is selected from the group consisting of: CNP, cGMP, type II collagen propeptide and fragment thereof, type II collagen and fragment thereof, type I collagen C-terminal peptide (CTx), osteocalcitonin, proliferating cell nuclear antigen (PCNA), type I collagen progenitor (PINP) propeptide and fragment thereof, type I collagen and fragment thereof, chondroitin sulfate, collagen X, alkaline phosphatase, proliferating cell nuclear antigen (PCNA), and type I collagen progenitor propeptide. The study included fragments of type I collagen, chondroitin sulfate, collagen X, CXM (non-collagen 1 (NC1) domain of type X collagen), NTproCNP, N-terminal type I collagen propeptide, bone-specific alkaline phosphatase, N-terminal propeptide of type I collagen / pro-N-propeptide of type I collagen (PINP), cross-linked type I collagen C-terminal peptide (CTx), cross-linked type I collagen N-terminal peptide (NTx) tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomic readouts, and CNP-variant bioactivity.

[0431] 72. The method of any one of Examples 63 to 70, wherein the administration increases the subject’s annualized growth rate (AGV) at 12 months, optionally compared to baseline or normal control.

[0432] 73. The method of Example 72, wherein the subject’s AGV increases over a period of 1 year or 2 years or longer.

[0433] 74. The method of any one of Examples 63 to 72, wherein the administration improves the height Z-score at 12 months, optionally compared to baseline or normal control.

[0434] 75. The method of any one of Examples 63 to 73, wherein the subject is older than 3 years.

[0435] 76. The method of any one of Examples 63 to 74, wherein the subject is between 3 and 17 years old.

[0436] 77. The method of any one of Examples 63 to 75, wherein the subject has an open epiphysis.

[0437] 78. The method of any one of Examples 63 to 77, wherein the composition is administered at a dose of about 5 μg / kg to 500 μg / kg or about 15 μg / kg to 350 μg / kg.

[0438] 79. The method of any one of Examples 63 to 77, wherein the administration does not cause cardiovascular (CV) side effects.

[0439] 80. The method of Example 79, wherein the CV side effects are changes in systemic blood pressure, mean arterial pressure, systolic and / or diastolic blood pressure, pulse pressure, or heart rate.

[0440] Example

[0441] In these examples and related diagrams, tables, figures, charts, etc., "free drug" is sometimes used to refer to CNPs that are not bound or conjugated to the connector and / or polymer.

[0442] Example 1: Synthesis of CNP variants

[0443] CNP variant peptides are synthesized on the solid phase using resins that leave C-terminal COOH on a Symphony / Prelude (Protein Technologies Inc., USA), Voyager (CEM GmbH, Germany) or Syroll (MultiSyntech, Germany) synthesizer.

[0444] All Fmoc-amino acids were purchased from Biosolve (Netherlands) or Bachem GmbH (Germany), and their side-chain functional groups were protected by Nf-Boc (KW), Of-Bu (DESTY), N-Trt (HNQ), S-Trt (C), or N-Pbf (R) groups. For each amino acid coupling step, double coupling was performed in NMP using a 5-fold excess of HBTU / HOBt / amino acid / DIPEA (1:1:1:2), with an activation time of 20 minutes.

[0445] Peptide acetylation (Ac) was performed by reacting the resin with NMP / Ac2O / DIEA (10:1:0.1, v / v / v) at room temperature for 30 minutes.

[0446] For a partial conjugation, the protective amino group on lysine is cleaved to produce a reactive group. Using standard Fmoc synthesis, 2× Fmoc-aminoPEG (2) is reacted, followed by glutamic acid, and then by C18-diacid.

[0447] The intact peptide was cleaved from the resin by reacting it with TFA (40 mL / mmol resin) for 2 hours at room temperature. The crude peptide was filtered, precipitated with ice-cold Et₂O, lyophilized, and finally purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The final product and purity were confirmed by mass spectrometry.

[0448] CNP variants are generated based on the sequence of Pro-Gly CNP-37 (PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1)), said CNP variants optionally having different amino acid residue variations and / or acetylation at the N-terminus, and / or other modifications, and including (amino acid variations underlined):

[0449] CNP-8: Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 8), CNP-5: Ac-PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 9); CNP-6: Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10);

[0450] CNP-7: Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 11); CNP-9: Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 12);

[0451] AC-PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-YGIU-C 18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 8) and

[0452] PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 7).

[0453] The fully reduced peptide was dissolved in 0.1 M Tris buffer (pH 8.0) with or without guanidine hydrochloride and containing 1 mM cysteine ​​(SS-form) and 8 mM cysteine ​​(SH-form) to a final concentration of 0.1 mg / mL, and stirred at room temperature. Disulfide formation was monitored by HPLC analysis until no further peak changes were observed. The mixture was then loaded onto a preparative RP-HPLC for purification.

[0454] Example 2: Characterization of synthesized CNP variants

[0455] Ten days later, the CNP variant synthesized as in Example 1 was analyzed by mass spectrometry and UV spectroscopy to determine its purity and stability.

[0456] Stability was measured using RP-HPLC purity analysis over time T0 to T10. In short, the CNP variant was diluted 1:5 in a pH 5.6 buffer: 5 mM citrate, 6% sucrose, 1.5% mannitol, 0.7 mg / ml methionine, and 0.005% Tween 80. A 10 μL injection volume was injected onto a Phenomenex Aeris XB-C18 RP column (2 μm, 100A, 2.1 × 250 mm; p / n 00G-4505-AN). HPLC conditions were: mobile phase A: H₂O / 0.05% TFA, pH 3, containing NH₄OH (final concentration approximately 3 mM); mobile phase B: 70% CH₃CN in H₂O / 0.05% TFA, pH 3, containing NH₄OH (final concentration approximately 3 mM); and a flow rate of 0.25 mL / min. Measurements were performed using a column temperature of 55°C and the following UV wavelengths: UV: 214 nm (bw 4 nm); ref 360 nm (bw 20 nm), UV: 280 nm (bw 4 nm); ref 360 nm (bw 20 nm). The CNP variants were incubated in PBS (pH 7.4) at 37°C for 10 days to obtain stability measurements.

[0457] The results of the stability measurements are shown in Table 1 below as the percentage of variants detected at T0 (0 days) or T10 (10 days).

[0458] Table 1

[0459]

[0460] The activity of CNP variants was tested using the CatchPoint Cyclic-GMP Fluorescent Assay Bulk kit (Molecular Devices, R8075) via cGMP stimulation assay. Specifically, NIH3T3 cells (ATCC, CRL-1658) and HEK293 cells were seeded at 60,000 cells per well in 96-well plates (96-well black imaging disc, Grenier, #655090). The culture media were as follows: NIH3T3 medium: DMEM high glucose, pyruvate (Thermo, 11995-073) + 10% FBS + 1× Pen Strep (abbreviated as P / S, Thermo, catalog number 15140122). NIH3T3 served as a control system for the HEK293 medium used in the cGMP assay: EMEM + 10% FBS + 1× P / S + 1× GMAX. For cells treated with IBMX (CAS 28822-58-4), serum-free NIH3T3 medium: DMEM + 1× P / S; for cells treated with CNP, serum-free NIH3T3 medium containing BSA: DMEM + 1× P / S + 0.5 mg / mL BSA (Thermo, A9418-100G).

[0461] Cells were cultured at 37°C and 5% CO2 for 24 hours. For cells intended to be treated with the CNP variant, each plate was pretreated with IBMX (Enzo Life Sciences, 89161-340, 1 g) 15 minutes before use. IBMX is a potent non-specific inhibitor of phosphodiesterase. 800 mM IBMX stock solution was diluted to 0.75 mM working stock solution in IBMX dilution medium (serum-free medium (DMEM + 1× PBS mixed with 1× PBS at a 1:1 ratio)).

[0462] CNP variants were prepared as follows: A 10 mg / mL CNP solution was diluted 1:1000 in CNP dilution medium (DMEM + 1×P / S + 0.5 mg / mL BSA). This solution was further diluted to obtain a starting CNP solution, and 100 nM CNP was plated per well. This 100 nM CNP solution was then serially diluted 1:5 six times to obtain the lowest final concentration of 0.0064 nM CNP per well. A 7-point dose-response curve was then provided for analysis.

[0463] For cell processing, remove cells from the incubator and remove growth medium from both the cells and those treated with IBMX. Add 80 μL of 0.75 mM IBMX to each well and return the cells to the 37°C incubator for 15 minutes. After 15 minutes, add 40 μL of CNP to each test well and return the cells to the 37°C incubator for 15 minutes. Mix the plate by gently tapping it. Image the plate on a Solentim cell metric to visualize the cells and determine if any cells have floated, then return it to the 37°C incubator.

[0464] Stop the reaction and lyse the cells by adding 40 μL of lysis buffer (from the cGMP kit). Place the plate on a shaker for 5 minutes to complete the lysis. Perform a cGMP assay using the cell lysate.

[0465] cGMP assays were performed using cGMP calibrator, rabbit anti-cGMP antibody, and HRP-cGMP prepared according to the manufacturer's protocol. 40 μL of calibrator was added to the wells of a plate coated with anti-cGMP antibody, and 40 μL of the dissolved product to be analyzed was added to the appropriate wells. 40 μL of reconstituted rabbit anti-cGMP antibody was added to all wells, and the plate was placed on a shaker for five minutes to mix. 40 μL of reconstituted HRP-cGMP was added to each well, and the plate was incubated at room temperature for 2 hours. The plate was manually aspirated and washed four times with 300 μL of wash buffer. 100 μL of stoplight red substrate was added to each well, covering the plate, and the plate was incubated at room temperature in the dark for at least 10 minutes. The fluorescence intensity of the plate was read on a Spectramax M or similar instrument at 530 nm excitation and 590 nm emission.

[0466] Table 1 shows the effect of CNP variants on cGMP production, indicating that the stable variants described in this paper can be used as therapeutic agents for bone-related conditions.

[0467] Example 2A: Stability of CNP variants or conjugates in plasma

[0468] The stability of different CNP variants or CNP conjugates in human plasma over a 24-hour period was tested. Briefly, two OEG spacers were used to fuse C18 fatty acids with γ-glutamic acid (see, for example, Lau et al., *Journal of Medicinal Chemistry* 58:7370-7380, 2015). For chromatography, Waters UPLC H-Class was connected to BEH C18 (1.7 μm, 2.1 × 150 mm); mobile phase A: 1% DMSO in water containing 0.1% formic acid; mobile phase B: 1% DMSO in CAN containing 0.1% formic acid. Mass spectrometry was performed using AB Sciex QTRAP. 200 nM of each CNP variant was prepared in lithium heparin from human plasma and sampled for incubation at 37 °C and 5% CO2. The reaction was quenched with 0.5 M sodium citrate (pH 4) at 0, 1, 2, 4, 8, and 24 hours. Plasma proteins were precipitated using MeOH containing 0.2% formic acid, and samples were prepared using WCX 96-well uElution plates and analyzed by LC-MS / MS 6500.

[0469] Variant CNP-R refers to a CNP37 variant in which a K residue not in the ring is replaced with an R residue, and CNP Q / R refers to a CNP37 variant in which an N residue is replaced with a Q residue and a K residue not in the ring is replaced with an R residue.

[0470] Figure 1 Figure 2 shows CNP conjugates with different linker structures. The esterified conjugates exhibit improved stability in plasma compared to PEGylated or unconjugated peptides in plasma.

[0471] The stability of different conjugates was also analyzed under different conditions. In short, the same protocols as described above were used at 37°C (control), 37°C + 0.5M NaCl, 37°C + protease inhibitor, or 4°C. Figure 3 The results showed that PG-CNP37 and its variants are readily hydrolyzed in human plasma, and that PG-CNP37 exhibits greater stability than other variants.

[0472] Example 3: Heterozygous NPR2 mutations respond to CNP treatment

[0473] To determine the effect of CNP on subjects with short stature caused by NPR2 mutations, a cell model of NPR2 mutations was developed. The exemplary NPR2 mutations analyzed are described in... Figure 6In this study, RCS cells with NPR2 gene knockout or heterozygous loss-of-function mutations were prepared by transfecting rat chondrosarcoma (RCS) cells with 125 ng of the NPR2 variant or by transfecting wild-type NPR2 plasmid DNA into RCS or HEK293 cells. Single-cell clones were seeded and genotyped by Sanger sequencing. The cell models were able to reproduce the cGMP phenotypes of the different mutations disclosed.

[0474] NPR2 clones were generated by creating insertions and deletions in the first exon of NPR2 in RCS cells. The sequence of the first exon of NPR2 was confirmed by next-generation sequencing and elucidated. Figure 5 In this study, the activity of NPR2 mutant cells in response to CNP administration was tested using cGMP stimulation assays via CatchPoint circular GMP fluorescence assay after treatment with 6 nM Pro-Gly CNP37. Rat chondrosarcoma (RCS) cells were seeded at 40,000 cells per well in RCS medium: DMEM + 10% FBS + 1× Pen Strep. Figure 4 Demonstrates cGMP readout of a rat chondrosarcoma cell model rescued with the addition of an exogenous Pro-Gly-CNP37 variant.

[0475] Previous activation data reported that for PRKG2 activation, the cGMP EC50 ranged from 40 to 360 nM (Campbell et al., ACS Chemical Biology 12, 2388-2398, 2017; Vaandrager et al., Journal of Biochemistry 272, 11816-23, 1997; Pohler et al., FEBS Lett 374, 419-25, 1995). In heterozygous NPR2 knockout cells, CNP doses >0.163 nM achieved intracellular concentrations exceeding the EC50 range of cGMP used for PRKG2 activation. Figure 4 In wild-type cells, a CNP dose of 0.040 nM achieved the same cGMP concentration. These results indicate that CNP supplementation can achieve the cGMP levels required for PRKG2 activation and growth in cells with NPR2 loss-of-function mutations.

[0476] These results also suggest that administration of CNP variants can be used to restore bone growth in short-statured subjects with reduced NPR2 activity. Further consideration suggests that treatment with CNP variants would be beneficial for subjects with mutations in other growth plate genes where cGMP signaling may be impaired.

[0477] Example 4: Identification of mutations associated with short stature

[0478] It is hypothesized that genes exhibiting clear evidence of bidirectional, gene-driven effects are more likely to represent therapeutic targets that can be effectively regulated across a broad patient population. To identify genes that are core regulators of growth, the intersection of five gene lists was analyzed, including lists from genome-wide association studies (GWAS). Core growth regulators will be highly likely to contain rare coding mutations with bidirectional effects (i.e., short stature or skeletal dysplasia versus tall stature or excessive growth).

[0479] The databases queried included: GWAS, which extracted 2,067 non-repeating closest genes for each of the 3,290 independent gene variants reported by a large GWAS height comprehensive analysis using approximately 700,000 individuals; HGMD, which queried the “allmut” table from HGMD version 2019_2 to find all pathogenic variants of the same gene marked “DM” that have “short stature” and “tall or overgrown”; and OMIM, a list of OMIM genes associated with growth disorders that had been previously described and created using the following keywords: short stature, overgrown, skeletal dysplasia, brachydactyly.

[0480] First, we searched the Human Genetic Mutation Database (HGMD v2019_2) for genes associated with short stature or tall stature (Stenson et al., Human Genetics 136:665-677, 2017). The literature reported 47 genes labeled with at least one pathogenic agent that cause short stature. Only 20 genes were labeled as genes for tall stature or excessive growth.

[0481] Second, a manually compiled list of 258 OMIM genes (248 for short stature and 20 for tall stature) was used, created using the keywords: short stature, excessive growth, skeletal dysplasia, brachydactyly (Wood et al., *Nature Genetics* 46:1173-86, 2014). Third, the intersection of these lists was compared with a gene list from GWAS. At the intersection of these lists, three known height-related genes were found (IGF1R, NPPC, NPR2), and two additional genes (FGFR3, SHOX) were identified.

[0482] Further analysis yielded a new set of five core genes showing a significant reduction in height (β = -0.20, 95% CI [-0.26 to -0.14], p = 4.04 × 10⁻¹¹) and a significant increase in the risk of idiopathic short stature (ISS) (OR = 2.75, 95% CI [1.92–3.96]). Each of the five core genes (FGFR3, IGF1R, NPPC, NPR2, and SHOX) was associated with height when considered alone and with short stature when combined with other mutations. Exemplary mutations in FGFR3, IGF1R, NPPC, NPR2, and SHOX are described in Figure 7 middle.

[0483] Combined loss of function (LoF) and missense variants in NPR2 and IGF1R were also associated with an increased risk of ISS (OR=3.31, P=0.001; OR=2.85, P=0.002, respectively). Whole gene deletions of SHOX, IGF1R, NPPC, and NPR2, and / or mutations therein causing loss of protein function, have been reported in familial short stature of varying severity. Mutations in DTL and pregnancy-associated plasma protein A2 (PAPPA2), or combinations thereof, also contribute to skeletal dysplasia.

[0484] Analysis showed that carriers of variants of any of the five core genes had an approximately 3-fold increased risk of ISS and accounted for 6.7% of the total ISS population. Furthermore, it was demonstrated that in a cell model with haplo-insufficient NPR2, the addition of exogenous CNP resulted in dose-dependent rescue of NPR2 signaling.

[0485] According to the omnigenic model (Liu et al., Cell 177:1022-1034e6 (2019); Boyle et al., Cell 169:1177-1186 (2017)), if these genes are core human growth genes, their functions should be regulated by multiple weaker, more common gene variants driving the regulatory network. To indirectly test this hypothesis, a polygenic risk score (PRS) for height was calculated using the published maximum GWAS meta-analysis for height, excluding any samples from the UK Biobank project. The cohort was divided into five equal-sized (n=6,824) PRS quintiles (PRS 1 being the shortest height and PRS 5 being the tallest). A dose-dependent relationship was found between the increase in PRS score and mean height (β=0.30 based on the increase per PRS quintile). Figure 8A Under five different PRS backgrounds, carriers of the LoF variant in the five core genes were consistently shorter than non-carriers. See Figure 8. The data suggest that the combined effect of PRS and rare protein variants is consistent with an additive model: polygenic effects regulate the height of carriers and non-carriers.

[0486] The risk of ISS was calculated for all PRS groups using PRS=3 as a reference. The lowest PRS group was associated with an increased risk of ISS, and the highest PRS group was associated with a decreased risk (OR=5.43, P=8.58×10⁻³⁴ for PRS 1 and OR=0.22, P=4.49×10⁻⁷ for PRS 5). The effect of rare coding variants of the five core genes on ISS stratified according to PRS group was assessed. In the top three quintiles, carriers of any of the five core genes had an increased risk of ISS (OR=2.64, P=3.09×10⁻⁵; OR=2.17, P=0.04; OR=5.29, P=1.58×10⁻⁵; OR=2.72, P=0.09). Figure 8C -F). For each individual core gene carrier, a consistent effect direction of ISS risk stratified according to PRS was observed ( Figure 8C -F).

[0487] Furthermore, the additive effect of PRS primarily stemmed from multiple common genetic variants with smaller individual effects, predicting a 20.1% variance in height across the dataset. These additive effects of PRS appeared to have similar magnitudes for both carriers and non-carriers of rare coding variants of the core gene. This observation suggests that PRS may be a significant factor contributing to differences in the penetrance of rare pathogenic variants (especially in haplo-insufficient models, such as those described in this paper). Supporting this idea, two out of eight NPR2 variant carriers with low NPR2 activity were observed to have shorter than normal heights. This data suggests that most individuals with NPR2 mutations in ISS may also have a polygenic background, making them more susceptible to pathogenic effects caused by loss of NPR2 activity.

[0488] These results support the view that CNP-based treatments can be effective in patients with insufficient NPR2 haplotypes. Furthermore, the results demonstrate a significant bidirectional (LoF and GoF) correlation between cGMP levels and height in NPR2 carriers in the general population, suggesting that targeting this receptor with CNP analogs could be an effective therapy for all individuals with short stature syndrome (ISS). A method for identifying variant genes associated with short stature (i.e., GoF or LoF variants) using reporter gene constructs / barcode assays is described in PCT / US24 / 32942, which is incorporated herein by reference.

[0489] Example 5: CNP formulation

[0490] Different buffers were used to assess particulate matter in CNP variant formulations. Specifically, particulate matter was identified by analyzing water-reconstituted lyophilized CNP formulations using visual inspection and UV concentration measurements. Figure 13 As shown in the diagram, visual inspection reveals the formation of visible particles in citrate-based formulations. Histidine buffer exhibits the best appearance among all the different buffers, including citrate.

[0491] As shown below, UV analysis highlights the decrease in concentration after precipitation.

[0492]

[0493] Example 6

[0494] Variant content was analyzed over time when stored for up to 13 months in different formulation buffers within a temperature range of -20 to 37°C. Results are shown in... Figure 14 middle.

[0495] Example 7

[0496] The levels of free drug released from different formulation buffers were analyzed over a period of time (up to 13 months) within a temperature range of -20 to 37°C. Results are shown in... Figure 15middle.

[0497] Example 8

[0498] The linker area of ​​the drug conjugates was analyzed over time when stored for up to 13 months in different formulation buffers within a temperature range of -20 to 37°C. Results are shown in... Figure 16 middle.

[0499] Example 9

[0500] The CNP conjugate content was analyzed over time when stored for up to 13 months in different formulation buffers within a temperature range of -20 to 37°C. Results are shown in... Figure 17 middle.

[0501] Example 10

[0502] Variant concentrations were analyzed over time by UV measurement when the products were stored in different formulation buffers for up to 3 months. Results are shown in... Figure 18 middle.

[0503] Example 11

[0504] Variants were analyzed over time (up to 13 months) in formulations using different excipients (histidine, glycine, and sorbitol), and pH 5.5 or 6 was assessed. Results are shown in... Figure 19 No precipitation was observed, and the excipients did not reduce drug cleavage. Histidine formulations showed no significant difference at pH 5.5 and 6.

[0505] Example 12

[0506] A manufacturing feasibility study was conducted on the CNP prodrug. The results are shown in… Figure 20 At most 0.5% free drug lysis was observed at 5°C and at most 2.5% free drug lysis was observed at 25°C. No significant difference was observed between pH 5.5 and pH 6.

[0507] Example 13

[0508] The degradation of CNP prodrug batches formulated with histidine or acetate buffer at different ratios (4:1 or 1:4) and trehalose and mannitol was analyzed over one month. Results are shown in... Figure 21 In the middle, the amount of free drug increases with time, temperature, and pH. Formulations containing dextran exhibit less free drug cleavage at pH 4, possibly due to the lower pH.

[0509] Example 14

[0510] Different CNP prodrug formulations were tested using buffer solutions, build-up agents, and other excipients.

[0511]

[0512] Example 15

[0513] As described above, additional analyses of the buffer were performed for precipitation at different time points and temperatures. The formulations evaluated included a CNP variant at 1 mg / ml in either 5 mM citrate (pH 5.5; 58 g / L trehalose, 15 g / L mannitol, 0.73 g / L L-methionine, 0.05 g / L PS80) or 10 mM citrate (pH 5.5; 58 g / L trehalose, 15 g / L mannitol, 0.73 g / L L-methionine, 0.05 g / L PS80). As shown below, precipitates were identified in the formulations containing citrate. No precipitates were identified in the formulations containing histidine.

[0514]

[0515]

[0516] Example 16

[0517] This produces additional CNP prodrug formulations.

[0518] Formulation buffer

[0519]

[0520] 10 mM histidine FB pH 5.5 and prodrug

[0521]

[0522]

[0523] Example 17

[0524] The area under the curve of the drug after reversed-phase chromatography was evaluated. The results are shown in... Figure 22 middle.

[0525] Example 18

[0526] The stability of a drug is assessed under the following conditions:

[0527]

[0528]

[0529]

[0530] Example 19

[0531] In-use stability and free drug concentration were assessed over time (up to 13 months) at temperatures ranging from 5 to 25°C and pH values ​​from 5.2 to 6.0. Results are shown in... Figure 23 middle.

[0532] Example 20

[0533] Describe a buffer formulation for CNP prodrugs.

[0534]

[0535] Antioxidants in the buffer solution are not essential, but they are added in some cases. L-methionine is particularly used as an antioxidant when employed.

[0536] PS 80 (polysorbate 80) is a preferred surfactant because it minimizes the loss of CNP variants caused by their absorption onto materials (e.g., containers for preparing formulations).

[0537] Example 21 – Visual Comparison of Different Amounts of Trehalose

[0538] Visual comparison of CNP prodrugs in different formulations is shown in Figure 24 A trehalose content of 3.5-5.5%, such as 3.8-4.8%, is acceptable. 4.8% trehalose is the preferred content because it exhibits advantageous properties upon visual inspection (and for other reasons).

[0539] Example 22 (Predictive) – Analytical techniques for analyzing the binding and / or linker cleavage and release of CNPs from conjugates in rat plasma.

[0540] Preparation of materials intended for analysis

[0541] For the purposes of this example, CNP refers to a CNP variant; in particular, it is assumed that the analysis will be performed using... Figure 10 The CNP variants shown herein are analyzed, but it is also envisioned that other CNP variants described herein can be analyzed using the same or similar methods.

[0542] 0.1M methionine

[0543] Weigh 60 mg of methionine. Add 4.0 mL of water to a 5 mL test tube. Mix thoroughly, seal tightly, and store on moist ice. Discard after use.

[0544] 0.5 M sodium citrate (pH 4.0)

[0545] Weigh 36.8 g of sodium citrate dihydrate. Dissolve it in approximately 220 mL of water. While stirring, adjust the pH to 4.0 using 1.0 M citric acid. Transfer the solution to a 250 mL volumetric flask and continue adding water until the meniscus reaches the graduation mark. Stopper the flask and invert it to thoroughly mix the solution.

[0546] Protease inhibitor mixture

[0547] 1) Dissolve the protease inhibitor mixture (Sigma P2714) in 10 mL of Milli-Q water to obtain inhibitor solution A.

[0548] 2) Dissolve 250 mg of 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor) (SigmaI5879) in 11.25 mL of methanol to obtain inhibitor solution B.

[0549] 3) Combine 0.83 mL of inhibitor solution B with 9.17 mL of inhibitor solution A to obtain 10.0 mL of protease inhibitor mixture. Store the remaining inhibitor solution B at -20°C for up to 2 months.

[0550] Treated rat plasma K2EDTA matrix

[0551] 1) Cool rat K2EDTA whole blood on ice and divide 10 mL into 15 mL test tubes.

[0552] 2) Add 2.0 mL of a mixture of 0.5 M sodium citrate (pH 4.0) and 60 μL of protease inhibitor to 10 mL of ice-cold rat K2EDTA whole blood.

[0553] 3) Mix gently, then centrifuge the 15 mL test tube at 1,500 × g for about 15 minutes at room temperature.

[0554] 4) Take the supernatant as the K2EDTA matrix for the treated rat plasma.

[0555] CNP stock solution

[0556] 10 mg of dried or lyophilized CNP was reconstituted in 1 mL of formulation buffer to provide a 10 mg / mL stock solution. The concentration was then corrected to approximately 2.3 mM based on the absorbance reading at 275 nm.

[0557] CNPox stock solution

[0558] 10 mg of CNP111ox was reconstituted in 1 mL of formulation buffer to obtain a 10 mg / mL Lip-BC-CNPox stock solution. After reading at A275 nm, the concentration was corrected to 8.64 mg / mL (2.10 mM).

[0559] Lip-BC-CNP stock solution

[0560] 10 mg of lipid-BC-CNP (MW: 4962.11) was reconstituted in 1 mL of formulation buffer to obtain a 10 mg / mL LiP-BC-CNP stock solution. After reading at A275 nm, the concentration was corrected to 6.88 mg / mL (1.38 mM).

[0561] Lip-BC-CNPox stock solution

[0562] 10 mg of lipid-BC-CNPox was reconstituted in 1 mL of formulation buffer to obtain a 10 mg / mL Lip-BC-CNPox stock solution. After reading at A275 nm, the concentration was corrected to 6.75 mg / mL (1.36 mM).

[0563] Mix ISTD stock solution

[0564] Prepare all ISWS on ice. Add 10 μL of 5 μM CNP (old) and 10 μL of 5 μM CNPox (old) to 5 mL of water containing 0.1% FA and 20% ACN. Vortex thoroughly, then aliquot the sample. Keep on ice. Add 10 mL of 5 mM Lip-BC-CNP^ (new) and 10 mL of 5 mM Lip-BC-CNPox^ (new) to 5 mL of formulation buffer. Vortex thoroughly, then aliquot the sample. Keep on ice.

[0565] standard solutions

[0566] The standard working solution was prepared using water containing 0.1% FA and 20% ACN as a diluent as follows:

[0567]

[0568] CNP and CNPox standards were prepared using treated rat plasma K2EDTA in 1.5 mL low protein binding tubes.

[0569]

[0570] Lip-BC-CNP and Lip-BC-CNPox standards were prepared using treated rat plasma K2EDTA in 1.5 mL low protein binding tubes.

[0571]

[0572] QC solution

[0573] The QC for preparing free peptide CNPs using citrate and inhibitor-treated rat plasma K2EDTA as a diluent is as follows:

[0574]

[0575] Lip-BC-CNP and Lip-BC-CNPox were prepared using citrate and inhibitor-treated rat plasma K2EDTA as diluents, with the following QC:

[0576]

[0577] LC-MS analysis conditions

[0578] Chromatographic conditions

[0579]

[0580]

[0581] Mass spectrometer parameters

[0582]

[0583] Example 22

[0584] Describe the buffer formulation for CNP variants.

[0585]

[0586] Example 23 – CNP prodrug active pharmaceutical ingredient and drug product

[0587] The following abbreviations are used in this article:

[0588]

[0589]

[0590]

[0591] Active pharmaceutical ingredient (DS)

[0592] CNP prodrugs contain CNP, a pH-responsive autolytic linker, a spacer, and a C-18 fatty acid albumin-binding domain (linked to Lys). 27(Side chain).

[0593] The sequence of the CNP prodrug is as follows: Pro 1 -Gly-Gln-Glu-His 5 -Pro-Asn-Ala-Arg-Lys 10 -Tyr-Lys-Gly-Ala-Asn 15 -Lys-Lys-Gly-Leu-Ser 20 -Lys-Gly-Cys-Phe-Gly 25 -Leu-Lys 27 (X)-Leu-Asp-Arg 30 -Ile-Gly-Ser-Met-Ser 35 -Gly-Leu-Gly-Cys-OH

[0594] (X) = OH-1,18-octadecanoyl-Glu(NH-PEG2-carboxyl)2-NH-CH2CH2-Gly(N-CH2CH2OH)-OH

[0595] The active pharmaceutical ingredient was isolated in hydrochloride form. Its empirical molecular formula is C0.05 217 H 363 N 61 O 65 S3 (in free base form). The average molecular weight is 4962.77 μm.

[0596] Physical and chemical properties

[0597] The relevant physicochemical properties of the available DS are provided in the table.

[0598] Table 3: Physicochemical properties of CNP prodrugs

[0599]

[0600] manufacture

[0601] The synthesis of CNP prodrug DS utilizes SPPS technology, employing Fmoc amino acid derivatives as structural units to assemble peptides onto a resin (solid support), and utilizing Fmoc-protected structural units for Lys 27 The externally assembled side chains (including pH-responsive self-degrading linkers, spacers, and C-18 fatty acid albumin-binding domains) were used. After synthesis, the peptide was cleaved from resin, cyclized, purified, and separated as a lyophilized powder. Descriptions of the synthetic methods are provided in Tables 4 and 5.

[0602] All starting material releases were performed in accordance with ICH Q7. These procedures are part of a standard quality program to ensure high peptide quality and batch-to-batch consistency.

[0603] Table 4: Manufacturing Flowchart of CNP Prodrug Active Pharmaceutical Ingredients

[0604]

[0605] 2-CTC, 2-chlorotriphenylmethyl chloride; SPPS, solid-phase peptide synthesis; TFA, trifluoroacetic acid.

[0606] Table 5: Description of Manufacturing Method

[0607]

[0608]

[0609] Characterization

[0610] The structure of the CNP prodrug has been described using various analytical techniques (Table).

[0611] Table 6: Structural Description of CNP Prodrugs

[0612]

[0613] Bioactivity

[0614] CNP prodrugs are inactive prodrugs of CNP. The linker hydrolyzes to form the active parent CNP, along with an inactive metabolite consisting of lipids and the hydrolyzed linker. The pharmacodynamics of the CNP prodrugs were evaluated in mice and NHP to confirm that the dose-response effect of CNP is preserved when the active CNP is continuously released into circulation from its albumin-bound inactive prodrug form compared to daily pulsed delivery of CNP. Completed studies demonstrate that treatment with CNP prodrugs preserves the expected dose-response endochondral growth effect of CNP, as evidenced by increased cell content and height in the hypertrophic zone of the growth plate and / or increased long bone length.

[0615] Preliminary specifications and batch analysis results of CNP prodrug active pharmaceutical ingredient

[0616] Control of residual solvents and elemental impurities will be based on ICH Q3C and ICH Q3D. Mandatory testing will be based on ICH Q6A. For clinical batch analysis, reference materials will be prepared and characterized for purity and impurity testing by reversed-phase ion-pair high-performance liquid chromatography-mass spectrometry. This paper provides preliminary batch release data.

[0617] CNP prodrug active pharmaceutical ingredient stability scheme

[0618] Stability was obtained for both non-GMP (non-clinical DS batches) and GMP (clinical DS batches) samples according to ICH Q1A conditions. Purity methods used in the analysis of stability samples will confirm stability as indicated by forced degradation studies performed according to ICH Q1A. Stability data were evaluated according to ICH Q1E to set a retest date under long-term storage conditions of -20±5°C. Additional stability data will be obtained under intermediate conditions of 5±3°C and accelerated conditions of 25±2°C / 60±5% relative humidity.

[0619] The stability profiles for CNP prodrug DS are provided in Tables 7-9.

[0620] Stability Data Overview

[0621] For non-GMP batches, representative stability data up to 9 months are currently available. Results show that DS remained stable for 9 months at -20±5℃ and 5±3℃. Stability studies conducted at 25±2℃ / 60%±5% RH showed increased impurities and moisture content after 6 months. However, based on these results, DS is expected to remain stable for at least 12 months.

[0622] Table 7: CNP prodrug active pharmaceutical ingredient stability protocol – Long-term (-20 ± 5℃)

[0623]

[0624] Table 8: Accelerated storage conditions for drug substance stability (5 ± 3℃)

[0625]

[0626] Table 9: Accelerated storage stabilization protocols for active pharmaceutical ingredients (25 ± 2℃ / 60% ± 5% RH)

[0627]

[0628] CNP prodrugs

[0629] CNP prodrug mixture

[0630] Table 10-1: Characteristics of Active Pharmaceutical Ingredients

[0631]

[0632] Table 10-2: Drugs without active ingredients

[0633]

[0634]

[0635] 1% (w / v) Polysorbate 80

[0636] The preparation of 1% (w / v) polysorbate 80 is described in Table 11 below.

[0637] Table 11: Preparation Instructions for 1% (w / v) Polysorbate 80

[0638]

[0639] Table 12: Instructions for preparing CNP prodrug preparation buffer

[0640]

[0641]

[0642] Target calculation of active pharmaceutical ingredients

[0643] Based on the required DP concentration and volume, the target amount of DS to be weighed is determined using the following formula:

[0644] Target calculation of active pharmaceutical ingredients

[0645]

[0646] Note:

[0647] -CNP variant hydrochloride, purity factor P=81.3%

[0648] -CNP equivalence factor B = 0.827

[0649] Figure 4 .1.2 is an example of determining the mass of DS (in milligrams) required to prepare 100 mL of 0.500 mg / mL DP solution.

[0650] Target calculation of active pharmaceutical ingredients

[0651]

[0652] Instructions for preparing drug solution

[0653]

[0654] program:

[0655] 1. Use Figure 4 The calculation in .1.1 determines the target quantity of DS.

[0656] 2. Weigh the DS quantity determined in step 1. Record the weight.

[0657] 3. Using the actual DS volume weighed in step 2 and the following formula, calculate the FB volume required to achieve the desired DP concentration:

[0658]

[0659] 4. Add 98% to 99% of the calculated CNP prodrug FB from step 3 to the pre-weighed DS from step 2, while reserving 1-2% of the FB for pH adjustment in subsequent steps.

[0660] Note: The amount of FB to be retained is based on the size; the larger the size, the larger the amount to retain.

[0661] 5. Mix until DS is completely dissolved and a homogeneous solution is obtained.

[0662] 6. Test and record the initial pH.

[0663] 7. Adjust the pH to 5.5 ± 0.1 using 1N sodium hydroxide. Record the volume used.

[0664] 8. Add an appropriate amount of CNP prodrug FB to the final volume, then mix gently until a homogeneous solution is obtained.

[0665] 9. Verify that the pH is 5.5 ± 0.1. Record the final pH.

[0666] Appearance: Clear, colorless liquid

[0667] Storage requirements / stability: In use: 25℃, not exceeding 6 hours;

[0668] Short-term: 2-8℃, not exceeding 3 days; and

[0669] Long-term: -60℃ or lower, not exceeding 1 month.

[0670] Description and composition of medicines

[0671] CNP prodrug DP is supplied as a preservative-free, lyophilized white to yellow powder, reconstituted with sterile water for injection (WFI). The reconstituted solution is colorless to yellow. Each vial contains 15.7 mg or 47.2 mg of CNP prodrug, equivalent to 13 mg or 39 mg of CNP, respectively. The reconstituted solution contains 10 mg / mL or 30 mg / mL of CNP equivalent at a target pH of 5.5. Sterile WFI will also be supplied for reconstitution. Clinical DP will be supplied in Type I single-dose sterile borosilicate glass vials with coated stoppers and flip-top aluminum caps. The formulation buffer contains 10 mM histidine buffer, 58.00 mg / mL trehalose dihydrate, 15.00 mg / mL D-mannitol, 0.73 mg / mL L-methionine, and 0.05 mg / mL polysorbate 80. It will be used as a placebo in clinical studies.

[0672] Table 13: Composition of CNP prodrugs

[0673]

[0674] Container closure system for CNP prodrug DP conforms to USP <660> and USP <381> (Table 14).

[0675] Table 14: CNP Prodrug Container Closure System

[0676]

[0677] Drug development

[0678] The proposed DPs for clinical use are representative of those used in animal toxicology studies that support the determination of the No Observed Adverse Effect Level (NOAEL) described herein. A description of the chemical and manufacturing differences between the DPs proposed for clinical use and those used in animal toxicology studies to determine the NOAEL is provided below.

[0679] Table 15: Overview of Drug Samples Used in Non-Clinical and Clinical Studies

[0680]

[0681] Formulation development began with a series of screening studies using different buffer species and at pH levels ranging from pH 3 to pH 8. Based on the results of these studies, histidine buffer (pH 5.5) was selected. pH range studies indicated the generation of specific impurities in the liquid phase, and lyophilization was chosen as the stabilization strategy. A series of studies were conducted using different build-up agents and their concentrations to develop isotonic and lyophilizable formulations for CNP prodrugs. Trehalose and mannitol in the amounts and ratios mentioned above (table) provide pharmaceutically aesthetically pleasing, pancake-like structures and isotonic formulations. L-methionine was added to the CNP prodrug formulation to reduce the likelihood of oxidation, while polysorbate-80 was included to minimize CNP prodrug adsorption to the product contact material.

[0682] Preliminary use studies were conducted, and no risks were detected. Compatibility studies were performed with the components of the described manufacturing method, and no risks were observed. Compatibility studies regarding the application of the components and the container's closed system are planned and will be used for IND submission.

[0683] In addition, method development studies were conducted, including the development and optimization of mixing, filtration, and lyophilization cycles, to support the manufacturing of the CNP prodrug DP. Using the results of these studies, development batches and engineered batches of the DP were manufactured to support GMP manufacturing. Both development and engineered batches demonstrated stability. Detailed data and stability data regarding these batches will be shared during the IND submission.

[0684] manufacture

[0685] The manufacturing process is illustrated in the following diagram and Table 16. A brief descriptive overview is also provided, including controls representing the combination of manufacturing process parameters, control tests, and quality attributes within the process.

[0686] CNP prodrug manufacturing process flowchart

[0687]

[0688] DP stands for Pharmaceuticals; DS stands for Active Pharmaceutical Ingredients; GMP stands for Good Manufacturing Practices.

[0689] Table 16: Descriptive Description of CNP Prodrug Manufacturing Methods

[0690]

[0691]

[0692] Careful control and monitoring of pharmaceutical manufacturing methods are essential to ensure consistency in the composition and quality of pharmaceutical products, thereby ensuring product safety and efficacy.

[0693] Drug specifications

[0694] The proposed DP specifications are provided in Table 17:

[0695] Table 17: Overview of Active Pharmaceutical Ingredient Batches

[0696]

[0697] Table 18: Batch Analysis of CNP Prodrug Active Pharmaceutical Ingredients

[0698]

[0699] Table 18 describes the specifications based on available product knowledge. All impurities >0.10% by area were released and stability monitored using the RP-UPLC purity method.

[0700] Drug stability

[0701] Ongoing stability studies were conducted on non-GMP engineered batches, while stability studies on GMP batches were conducted post-manufacturing. Stability studies were performed using principles from ICH Q1A, and stability data were assessed using principles according to ICH Q1E. The stability protocol for CNP prodrug DPs is provided in Table 18 and below. Stability data obtained from engineered batches are expected to support the stability of clinical DPs throughout the duration of clinical studies. DP process testing provides adequate control over the methods used for both engineered and clinical batches. There were no differences in manufacturing methods or container closure between engineered and GMP batches.

[0702] Table 19: CNP prodrug stability regimen – Long-term (5 ± 3℃)

[0703]

[0704]

[0705] Table 20: Accelerated storage conditions for drug stability (25 ± 2℃ / 60 ± 5%RH)

[0706]

[0707]

[0708] Manufacturing method

[0709] This example demonstrates a preparation method, described below, for manufacturing a CNP prodrug, the method comprising linear and side-chain assembly via solid-phase synthesis (SPPS), TFA cleavage / deprotection, in-solution cyclization using I2, reversed-phase chromatography (RPC) purification, and salt exchange:

[0710] Artificial solid-phase synthesis (SPPS)

[0711] Artificial SPPS of CNP prodrugs were performed on chlorotriphenylmethyl chloride (CTC) resin. The table below describes the coupling time, amino acid equivalents, Fmoc deprotection time, and AA residue acetylation required during chain amplification.

[0712] Table 21. SPPS of CNP prodrugs

[0713]

[0714]

[0715] General coupling schemes:

[0716] ● Dissolve AA:DIC:Oxymapure (1:1.1:1) and AA in DMF to achieve a concentration of 0.2-0.3 M.

[0717] ● Add 0.1 M Oxymapure to the Fmoc removal step to minimize the racemization of Cys during alkaline treatment.

[0718] ● For AA coupling solutions other than Arg, Cys, His and Met, pre-activate for 0.5-1 hour.

[0719] ● Before side chain assembly, side chain deprotection is performed at A-11 using Fmoc-Lys(Alloc)-OH.

[0720] ● Use Pd(PPh3)4 / PPh3 / DMBA (0.1eq:0.5eq:5eq in DMF) to remove Alloc

[0721] ● Use DEPBT for His coupling to suppress racemization (His:DEPBT:DIPEA = 2:2.2:3-3.5, approximately pH 9)

[0722] ● Sidechain assembly is performed using a single AA coupling strategy.

[0723] ● Apply N2 coverage throughout the SPPS to minimize Cys and Met oxidation.

[0724] 1. Pyrolysis and Cycloning

[0725] For pyrolysis, the following conditions were used: TFA: DTT: H2O: TIPS: NH4I = 87:7.5:2.5:2.5:0.5, 3 hours, room temperature, nitrogen atmosphere. Then, reverse precipitation was performed by adding the pyrolysis mixture (cooled to 1-15°C) to 4V of 25% n-heptane / MTBE (pre-cooled to 4°C) over 15 minutes.

[0726] Next, the crude lumps were dissolved in HOAc + 20% ACN at 30 mg / mL and kept for 30 minutes. ACN containing 2% I2 was added to initiate oxidation until the color remained orange. After this, the oxidation was quenched with 1% ascorbic acid / H2O.

[0727] 2. Purification

[0728] In addition to the first reverse-phase purification performed on C18 medium (A: 0.1M TEAP (pH 2.3) in H2O, B: 0.1M TEAP (pH 2.3) in ACN, 25-45% B after 100 min), a second purification / salt exchange was performed to convert the TFA salt to hydrochloride medium (A: 0.02% HCl in H2O, B: 0.02% HCl in ACN, 10-15% B after 3 min, 15-50% B after 75 min).

[0729] 3. Freeze-drying

[0730] The final pooled samples were filtered through a 0.2 μm filter and then freeze-dried. Typical yield: 6 to 7.5%.

[0731] Manufacturing process

[0732] Active pharmaceutical ingredient – ​​Additional information

[0733] The CNP prodrug peptide was synthesized using solid-phase peptide synthesis (SPPS) technology. Na-9-fluorenylmethoxycarbonyl (Fmoc) amino acid derivatives were used as structural units to assemble the peptide onto a resin (solid support), and side chains were assembled outside Lys 27 using Fmoc-protected structural units. After synthesis, the CNP prodrug active pharmaceutical ingredient (DS) was cleaved from the resin, cyclized, purified, and separated into a lyophilized powder.

[0734] The specifications for release and stability testing will conform to the general principles of the International Council for Harmonisation (ICH) Q6A for the technical requirements of the new DS.

[0735] The proposed acceptance criteria for the purity of clinical batches are set at ≥95% area and total impurities (≤5%).

[0736] The acceptance criteria for residual solvents (i.e., ICH Q3C), elemental impurities (i.e., ICH Q3D, and including other elements only for monitoring purposes) were developed with reference to relevant ICH guidelines and United States Pharmacopeia (USP) / European Pharmacopeia (Ph. Eur.) monographs.

[0737] The endotoxin limitation of CNP prodrug DS is based on USP. <85> This complies with the provisions of Chapter 5.1.10 of the European Pharmacopoeia regarding parenteral products administered intravenously (IV). Endotoxin levels of CNP prodrugs (DPs) will also be controlled based on clinical dosage and route of administration to ensure product safety. Acceptance criteria for bioburden follow USP. <61> .

[0738] In summary, the selected test parameters and release acceptance criteria were deemed appropriate to ensure the correct identity, suitable chemical / microbiological purity, and reliable assay values ​​of the CNP prodrug DS at this stage of clinical development.

[0739] Medications – Additional Information

[0740] Test parameters and acceptance criteria for the release and shelf life of CNP prodrug DPs were selected to confirm their identity, purity, quality, and ensure product safety. Acceptance criteria have been established and adjusted based on limited manufacturing experience and general principles of ICH Q6A. The relative residence time and area percentage of all impurities ≥0.10% will be reported and monitored during release and stability testing. Testing for pH, sterility, endotoxin, osmolality, container contents, particulate matter, and container closure integrity will be conducted according to ICH Q6A guidelines for parenteral products, and acceptance criteria will be determined based on all applicable guidance documents, relevant USP sections for sterile products, and current manufacturing understanding.

[0741] Table 23: Overview of Active Pharmaceutical Ingredient Batches

[0742]

[0743]

[0744] Table 24: Batch Analysis of CNP Prodrug API

[0745]

[0746] Table 25: Preliminary Description of CNP Prodrugs

[0747]

[0748]

[0749] Table 26: Overview of Non-Clinical Studies of CNP Prodrugs

[0750]

[0751] Metabolites

[0752] CNP prodrugs include those via Lys 27The CNP has a side chain that binds to a pH-responsive, self-degrading linker, a spacer, and a C-18 fatty acid albumin-binding domain. Upon hydrolysis of the linker, an active CNP and two major pharmacologically inactive metabolite isoforms are formed. These isoforms consist of an albumin-bound C-18 fatty acid and a hydrolyzed linker, and are designated metabolite A and metabolite B, respectively. The structures of the two inactive metabolite isoforms are shown below.

[0753] Structure of metabolite A

[0754]

[0755] Structure of metabolite B

[0756]

[0757] Metabolites A and B are expected to co-form in NHP in a similar proportion to that of the CNP prodrug in humans, as the hydrolysis rate of the CNP prodrug is constant at physiological pH. Therefore, the toxicity of each metabolite isoform will be assessed as part of the general toxicity study of the CNP prodrug. Metabolite A is unstable and rapidly forms metabolite B via hydrolysis during synthesis and purification; therefore, metabolite B is the predominant metabolite isoform likely to be detected in the plasma of both humans and non-clinical species. Two computer simulation methods will be used to assess the two metabolite isoforms.

[0758] Since there are no plans to conduct genotoxicology studies of the CNP prodrug, metabolite B will be further evaluated in in vitro bacterial reverse mutation assays and in vitro micronucleus assays. Metabolite A is too unstable to be produced because it spontaneously forms metabolite B, and therefore cannot be evaluated in genotoxicology studies.

[0759] Where appropriate, additional metabolite identification and characterization of metabolite toxicity, absorption, distribution, metabolism, and excretion properties will be conducted in accordance with the FDA’s guidance on Safety Testing of Drug Metabolites (March 2020).

[0760] The inhibitory effects of CNP prodrug or metabolite B on CYP enzymes (e.g., CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 / 5 enzymes) in human liver microsomes were evaluated in vitro. CNP prodrug and metabolite B did not inhibit the activity of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 / 5 in vitro at concentrations up to 20 μM. No direct, time-dependent (30 min pre-incubation), or metabolism-dependent (30 min pre-incubation with NADPH) inhibition of CNP prodrug or metabolite B on CYP enzyme activity was observed for each tested CYP. The inhibitory effects of CNP prodrug or metabolite B on CYP enzymes (e.g., CYP1A2, CYP2D6, and CYP3A4 enzymes) in cultured human hepatocytes were continuously evaluated.

[0761] Metabolites A and B were screened using computer simulations employing quantitative structure-activity relationship (QSAR) assessment. The computer simulation analysis did not identify any risks associated with CNP prodrug administration. Based on QSAR analysis, neither metabolites A nor B is likely to be mutagenic.

[0762] Evaluation of CNP prodrugs and lipid adaptor substrates for in vitro transport in different cell lines is underway.

[0763] Phase 1 Study

[0764] BioMarin plans to initiate a comprehensive clinical development program, including a Phase 1 study in healthy adult volunteers, followed by a Phase 2 dose range discovery and a pivotal Phase 3, to support approval of the CNP prodrug in pediatric patients with ACH.

[0765] Example 24: Pharmacodynamics of CNP prodrugs

[0766] CNP prodrugs are pharmacologically inactive prodrugs of CNP that do not bind to NPR-B (internal data). The linker hydrolyzes to form the active parent CNP, as well as an inactive metabolite isoform consisting of lipids and the hydrolyzed linker. Therefore, the pharmacodynamics of CNP prodrugs depends on the pharmacological activity of the released CNP. CNP has a defined mechanism of action and has been shown to increase intrachondral bone growth in animal models of WT and FGFR3-mediated chondrodysplasia, and is approved for the treatment of ACH in children with open epiphyses. Therefore, pharmacological studies of CNP prodrugs are designed to confirm the desired pharmacological action of CNP retained from the CNP released from the prodrug and to determine that sustained CNP exposure is required to achieve intrachondral bone growth.

[0767] The pharmacodynamics of CNP prodrugs were evaluated in mice and NHP to demonstrate that the dose-response effect of CNP is preserved when active CNP is continuously released into circulation from its albumin-bound inactive prodrug form, compared to daily pulsed delivery of CNP. The completed studies confirmed that treatment with CNP prodrugs preserves the expected dose-response endochondral growth effect of CNP, as evidenced by increased cell content and height in the hypertrophic zone of the growth plate and / or increased long bone length.

[0768] In a study entitled "Five Weeks Growth Study of CNP Prodrug Compared to CNP in C57BL / 6J Mice," the growth effects of CNP prodrug in young WT mice were compared with those of CNP. A total of 80 three-week-old male C57BL / 6J mice (Jackson Laboratories) were randomly assigned to seven treatment groups. At the start of the study, the animals weighed 6.3 to 7.2 g. Animals were treated for 5 weeks by daily SC injection of 0 (mediator), 40, 150, or 500 μg / kg CNP, or by every other day (QAD) SC injection of 0 (mediator), 80, 300, or 1000 μg / kg CNP. All doses were based on CNP content. The study design is shown in Table 24-1.

[0769] Table 24-1: Design of pharmacodynamic studies of CNP prodrugs in young wild-type mice

[0770]

[0771] General health status of the animals was observed daily. Body weight was collected before each administration (daily for groups A, C, D, and E; and QAD for groups B, F, G, and H). Overall growth was assessed weekly by measuring nose-anus and tail length using a ruler while the mice were anesthetized with inhaled isoflurane, beginning before treatment. Femur, tibia, humerus, ulna, and spine (upper cervical to lower sacral vertebrae) were measured weekly based on micro-computed tomography (μCT) images performed during inhaled isoflurane anesthesia. The study veterinarian also reviewed evidence of abnormalities in the μCT images. Animals were euthanized and necropsy performed after 5 weeks of treatment.

[0772] When baseline differences were taken into account, there was no effect on weight gain. No changes in gait or walking were observed, or any gross abnormalities were observed at necropsy. Compared to mice treated with the same mediator, a significantly increased dose-responsiveness in nose-anal length was observed in all CNP-treated mice. As shown in Table 25, a significant increase in nose-anal length was observed only at 1000 μg / kg QAD in mice treated with CNP prodrug. Compared to the parallel control, tail length in CNP-treated mice increased significantly only at doses of 150 μg / kg and 500 μg / kg daily. No effect on tail length was observed in mice treated with CNP prodrug.

[0773] Table 24-2: Growth of nose-anus and tail length in young WT mice treated with CNP and treated with CNP prodrug

[0774]

[0775]

[0776] Estimation of prodrug and CNP C max The values ​​and AUC are listed in Table 24-2.1.

[0777] Table 24-2.1: Estimation of prodrug and CNP max and AUC

[0778]

[0779] The levels of cGMP, a biomarker for CNP binding to the NPR-B receptor, were measured in plasma collected at 0.5, 4, and 24 hours post-dose. Although plasma cGMP levels increased at 0.5 hours post-dose in CNP-treated mice at all doses, cGMP levels at 4 hours post-dose were similar to those in mice treated with the carrier agent. In contrast, with the CNP prodrug, sustained release of cGMP was observed at all time points at a dose level of 1000 μg / kg QAD. cGMP levels were approximately one-quarter of the peak observed with CNP treatment when compared to those in CNP-treated mice. cGMP was undetectable in the plasma of mice treated with the CNP prodrug at 80 or 300 μg / kg QAD.

[0780] Although significant dose-responsive growth of the femur, tibia, and spine was observed in CNP-treated mice, no significant growth was observed in mice treated with CNP prodrugs. This study demonstrates the pharmacological activity of treatment with 1000 μg / kg QAD CNP prodrugs in WT mice, with effects on nose-anal length and detectable plasma cGMP. Nevertheless, it is concluded that QAD administration of CNP prodrugs does not allow for sufficiently constant CNP exposure in mice to produce growth effects similar to those produced by daily CNP administration.

[0781] Growth effect of daily administration of CNP prodrug in WT mice

[0782] The effect of increasing the (daily) administration frequency of CNP prodrug was evaluated in a study entitled “A 5 Week Study of CNP and CNP Prodrug by Subcutaneous Administration in Juvenile Male Mice”.

[0783] On day 21 after birth (PND 21), a total of 40 male C57BL / 6NCrl mice (Charles River Laboratories) were randomly assigned to four treatment groups. Animals were treated with daily subcutaneous injections of a carrier, 500 μg / kg CNP daily, or 500 or 1600 μg / kg CNP prodrug daily. All doses were based on CNP content. All animals were administered medication starting on PND 22, and treatment was intended to last for 5 weeks (until PND 57); however, treatment was prematurely terminated after 18 days due to the test substance effect. The study design is shown in Table 24-3.

[0784] Table 24-3: Design of a pharmacodynamic study involving daily administration of CNP prodrug to young wild-type mice.

[0785]

[0786] Animal mortality, clinical observations, and body weight were monitored. Digital radiographs were acquired under general anesthesia on PND 21 (pre-test), PND 29 (study day 8), and PND 36 (study day 15). Additionally, radiographs were acquired on PND 41 (study day 20) of animals exhibiting suspected fractures (including the remaining controls). At the scheduled necropsy, tissues of the right femur, right humerus, and right ulna were trimmed, and length and width were measured using calipers.

[0787] Several animals in the 500 μg / kg CNP daily treatment group (Group 2) and the 1600 μg / kg CNP prodrug daily treatment group (Group 4) were euthanized early. On day 4, a group of four animals were euthanized due to adverse / deteriorating conditions. Given the short duration of treatment, this death was considered likely related to maladaptive weaning rather than to CNP prodrug treatment. On day 8, a group of four animals were euthanized due to slight limitation of use of the right forelimb, suspected to be a result of cage-related injury; the relationship to CNP prodrug treatment was uncertain due to the short duration of treatment. On days 15–16, four out of ten animals in Group 2 and five out of eight remaining animals in Group 4 were euthanized due to suspected fractures and / or mild to moderate stiffness and swelling of both hind limbs and abnormal gait. Due to these early deaths, the study was terminated on day 20, and all surviving animals were euthanized and post-mortem examined.

[0788] During the administration period, at 500 μg / kg daily, bilateral CNP-related radiographic findings were observed in the distal tibia and calcaneus of male animals. On day 8 of the study, all animals showed radiographically increased epiphyseal plate thickness and a mixed radiographic response in the distal tibia. On day 15 of the study, radiographically increased epiphyseal plate thickness and a mixed radiographic response in the distal tibia, as well as increased epiphyseal plate thickness in the calcaneus and suspected or confirmed distal tibial fractures, were still observed in some animals. Additionally, sporadic epiphyseal fractures of the calcaneus were present. On day 20 of the study, all remaining male animals showed radiographically increased epiphyseal plate thickness and a mixed radiographic response in the distal tibia, as well as suspected or confirmed distal tibial fractures. Most male animals showed epiphyseal fractures of the calcaneus and / or increased epiphyseal plate thickness. Radiographic findings are summarized in Table 24.4.

[0789] During administration, at ≥500 μg / kg / day, bilateral CNP prodrug-related radiographic findings were observed in the distal tibia and calcaneus of male animals, generally similar to those observed with CNP. On day 8, all male animals at 1600 μg / kg / day showed radiographic increases in epiphyseal plate thickness and mixed radiographic responses in the distal tibia. On day 15, increased epiphyseal plate thickness and mixed radiographic responses in the distal tibia were commonly observed in animals treated with 500 or 1600 μg / kg CNP prodrug or 500 μg / kg CNP daily. These findings were associated with increased calcaneal epiphyseal plate thickness and suspected or confirmed distal tibial fractures in some animals treated with 500 μg / kg CNP or 1600 μg / kg CNP prodrug daily. In addition, one male animal treated daily with 500 μg / kg CNP prodrug presented with a calcaneal epiphyseal fracture. On day 20 of the study, prior to necropsy, radiography was performed only on animals clinically suspected of fracture (3 / 6 of the animals treated with 500 μg / kg CNP and 1 / 6 of the animals treated with 500 μg / kg CNP prodrug) and two representative control animals. Similar to days 8 and 15, increased epiphyseal plate thickness and mixed responses (areas of both increased and decreased bone density) were observed on radiographs of the distal tibia and calcaneus. Calcaneal epiphyseal fractures were observed in the two CNP-treated animals and the animals treated daily with 500 μg / kg CNP prodrug. Radiographic findings are summarized in Table 24-4.

[0790] Table 24-4: Relevant radiographic findings of test samples on days 8, 15, and 20 of the study.

[0791]

[0792]

[0793] Radiographic bone measurements were performed on days 8 and 15 of the study. Although increased epiphyseal plate thickness was observed in radiographs of mice with fractures, no treatment-related effects on the length of the femur, tibia, ulna, humerus, or lumbar vertebrae were observed at any time point compared to controls in mice treated with 500 μg / kg CNP prodrug daily or with CNP alone. On day 15 of the study, animals treated with 1600 μg / kg CNP daily had significantly greater femur (+11%) and lumbar vertebrae (+12%) lengths compared to controls. No other CNP prodrug-related effects on the length of the tibia, ulna, or humerus were observed. The interpretation of the terminal ex vivo measurements was affected by the reduced number of surviving animals in the 500 μg / kg CNP daily treatment group and the 1600 μg / kg CNP prodrug daily treatment group. At the end of the dosing period, compared with the control, male animals administered 500 μg / kg CNP prodrug daily showed statistically significant increases in femur length (+7%), humerus length (+4%), and ulna length (+7%); however, high inter-animal variability suggests that these findings may be accidental. No therapeutic effect was detected in histomorphological assays, but this endpoint was affected by the number of early deaths and the reduced number of animals surviving to day 20 of the study.

[0794] Microscopic findings in animals euthanized on or after day 15 of the study are detailed in Table 24.5. Mild to severe increases in distal tibial epiphyseal plate thickness were observed at daily doses of 500 μg / kg CNP and 500 or 1600 μg / kg CNP prodrugs, associated with grossly visible joint swelling. Epiphyseal plate thickness increased in most animals, progressing from mild to significant epiphyseal plate degeneration / necrosis, and epiphyseal plate fractures occurred in a minority of animals, primarily in the 1600 μg / kg CNP prodrug group. Epiphyseal plate fractures were associated with clinically observed joint swelling. At the same dose, the increase in distal tibial epiphyseal plate thickness was accompanied by two secondary changes. A slight to mild increase in metaphyseal bone, considered as primary and secondary elongation of cancellous bone, and a slight to mild increase in cortical resorption, considered as elongation of the "cut-back" zone.

[0795] In the proximal tibia, the most significant change was a minimal decrease in epiphyseal plate thickness, observed with daily administration of 500 μg / kg CNP or CNP prodrug, and more prevalent in animals treated with CNP prodrug. This change was associated with a reduction in epiphyseal plate parameters measured by histomorphology. A minimal increase in epiphyseal plate thickness was observed in a few animals at ≥500 μg / kg CNP prodrug daily, and this change was dose-dependent. This change was accompanied by a minimal increase in bone density in the metaphysis of an animal treated with 1600 μg / kg CNP prodrug daily, which was considered a secondary change. Minimal chondrocyte vacuolation was observed at 500 μg / kg CNP daily. This was characterized by chondrocyte vacuolation in hypertrophic areas.

[0796] In the calcaneus, the changes are similar to those described in the distal tibia and consist of a mild to moderate increase in epiphyseal plate thickness, mild to moderate epiphyseal plate degeneration / necrosis, fracture, and mild to moderate cortical resorption (all observed on the dorsal surface of the bone). Fracture is associated with visible joint swelling.

[0797] Table 24-5: Microscopic findings related to test substances in wild-type mice

[0798]

[0799]

[0800] In summary, increasing the dose interval from QAD to daily SC administration of the CNP prodrug resulted in increased plasma CNP levels, leading to adverse effects on the tibia and calcaneus of juvenile WT mice at doses of 500 or 1600 μg / kg daily. Similar effects were observed with high daily doses of CNP (500 μg / kg daily). These changes reflect previously observed exaggerated pharmacological effects of CNP in rodents, sometimes with no or moderate effects on bone length. Despite these effects, the lack of significant growth effects in this study should be considered in the context of the short treatment duration and detectable subtle growth effects. As discussed below, this fracture effect was not radiographically observed via μCT in WT mice of different strains treated daily with 500 μg / kg CNP or 1600 μg / kg CNP prodrug for 6 weeks.

[0801] Example 25: Noonan syndrome mouse model

[0802] Noonan syndrome (NS) is a genetic disorder affecting MAPK signaling pathways in multiple cell and tissue types, and causing varying degrees of cardiac and bone morphology disruption. Clinical manifestations of NS pathobiology include decreased height and moderate facial skeletal changes, as well as hypertrophic cardiomyopathy (HCM). The genetic etiology of NS involves autosomal dominant mutations in key signaling components of the RAS signaling cascade, such as protein tyrosine phosphatase non-receptor type 11 (RIT1), and rapidly accelerating fibrosarcoma 1 (RAF-1) (Saint-Laurent 2024, *European Journal of Pediatrics*; 183:1011-1019). Mutations in these signaling intermediates reduce the negative regulatory pathways required for normal ERK / MAPK homeostasis, leading to increased MAPK activity. As in ACH, this increase in MAPK signaling can be reduced by treatment with CNP or CNP antagonizes MAPK signaling at RAF-1 levels. Therefore, these models can be used to demonstrate the efficacy of CNP prodrugs versus CNPs in cases of increased MAPK signaling.

[0803] This study, titled "Pilot Pharmacodynamic Study of CNP and CNP Prodrug in Juvenile Wild Type or Raf1 L613V Heterozygous Male Mice Dosed Daily via Subcutaneous Administration for 6 Weeks," aimed to evaluate the effects of CNP and CNP prodrug on growth in the presence of Raf1 L613V activating mutations. These effects were compared to the small molecule MAPK kinase inhibitor (MEKi) PD0235901, which had previously been evaluated in this model (Wu 2011, J Clin Invest 121:1009-1025).

[0804] Raf1 + / L613VMice expressed a wild-type copy of the Raf1 gene and a copy containing a gene knock-in missense mutation in which leucine at residue 613 was replaced with valine (L613V). This mutation caused overactivation of the MAPK pathway. Compared to wild-type sibling mice, heterozygous mice carrying this mutation began to develop phenotypes similar to those observed in NS patients at 5 weeks of age, including cardiac enlargement, mild flattening of craniofacial features, and mild shortening of the naso-anal length (Wu, see above).

[0805] In this study, by using male Raf1 + / L613V Male WT or Raf1 mice with a 50% 129S1 / SvlmJ background and a 50% C57BL / 6J background were bred by crossing 129S1 / SvlmJ mice (customized via clustered regularly spaced short palindromic repeats [CRISPR]Cas9 technology; Jackson Laboratories, Sacramento, CA) with female WTC57BL6 / J mice (JAX stock #000664, Jackson Laboratories, Sacramento, CA). + / L613V Mice. Genotyping of animals was performed at PND 7 by clipping the tail tip. A total of 25 WT male mice and 38 Raf1 mice were weaned at 3 weeks of age (PND 21-23). + / L613V Male mice (8-12 g weight) were divided into 8 groups. The study design is shown in Table 25-1.

[0806] Table 25-1: In WT mice and Raf1 + / L613V Design of pharmacodynamic studies in Noonan syndrome mice

[0807]

[0808] After randomization, animals were anesthetized and underwent baseline echocardiography, weight measurement, and nose-to-anus length measurement using a standard metric ruler. At 3 weeks of age, Raf1... + / L613V They were approximately 0.46 cm or 6.72% shorter than age-matched WT littermates and had correspondingly lower body weights. Cardiac hypertrophy was present in this model, but the HCM phenotype was not observed at baseline and did not develop with or without CNP or CNP prodrug treatment.

[0809] Treatment began at 4 weeks of age. Wild-type mice and Raf1 mice were treated with daily SC injections. + / L613V Mice were administered a mediator, 500 μg / kg CNP daily, or 1600 μg / kg CNP prodrug daily. All dosages were based on CNP content. Raf1+ / L613V The mouse control group received intraperitoneal (IP) injections of either the carrier or 5 mg / kg MEKi daily. All animals were administered the medication for 6 weeks.

[0810] Animals were examined daily for physical condition, behavior, and gait, and weighed daily before administration of medication. During week 5 of the study, nose-to-anus length was measured, and echocardiography was performed under anesthesia. Mice in the AF group underwent whole-body μCT scans to assess femur and tibia length and skull morphology before euthanasia in week 6. Animals were euthanized and necropsy performed on days 49–52 (week 6 of the study). Right femurs and tibias were collected from all WT animals. Hearts and spleens were weighed.

[0811] There were no test-related deaths. One animal in group H (5 mg / kg MEKi daily) failed to recover from anesthesia after a cardiac ultrasound procedure at week 5 of the study. Two weeks after treatment, animals treated with CNP or CNP prodrugs began to develop significantly elongated skeletal features and mild peripheral bone growth, resulting in kyphosis and paw curling. Mice treated with MEKi rapidly gained weight and developed mild obesity by week 5 of the study.

[0812] As shown in Figure 25, at 8 weeks of age, Raf1 was observed at 3 weeks of age. + / L613V The reduced nose-anus length phenotype in mice no longer existed because Raf1 mice treated with the catalytic agent... + / L613V The length of the mice was not significantly different from that of the WT animals treated with the carcass. Compared with the carcass-treated mice, wild-type mice treated daily with 1600 μg / kg CNP prodrug showed a 21.2% increase in nose-to-anus length and Raf1 + / L613V The nose-anal length of mice increased by 12.4%. Compared with mice treated with carcasses, wild-type mice treated daily with 500 μg / kg CNP showed a 7.8% increase in nose-anal length, and Raf1... + / L613V Mice length increased by 7.4%. Compared with mice treated with 500 μg / kg CNP daily, WT mice and Raf1 mice treated with CNP prodrug showed [significant improvement]. + / L613V It exhibits a significantly longer naso-anal length. Treatment with mitogen-activated protein kinase inhibitors was also associated with an increase in naso-anal length compared to the mediator control, but these increases were smaller than those observed with CNP or CNP prodrug treatment.

[0813] At the end of the 6-week treatment period, no fractures were detected by μCT, contrasting with effects observed in C57BL / 6NCrl mice. Similar to the nose-anal length, by 9 weeks of age, mediator-treated Raf1 mice showed no fractures by μCT. + / L613VThere was no difference in femoral or tibia length between mice treated with the causative agent and WT mice. Statistically significant growth in long bones was induced by daily CNP prodrug (1600 μg / kg) and daily CNP (500 μg / kg) in WT mice (Table 25-2). Statistically different growth effects were also observed between CNP prodrug and CNP in WT mice, with CNP prodrug at 1600 μg / kg daily inducing greater femoral and tibia growth than CNP at 500 μg / kg daily (p<0.0001, determined by two-way ANOVA and Durkheim's multiple comparison test).

[0814] Table 25-2: WT and Raf1 after 6 weeks of treatment + / L613V Femur and tibia length in mice

[0815]

[0816] At 9 weeks of age, compared with WT sibling mice, Raf1 + / L613V The mice exhibited a mild increase in medial canthal distance, a decrease in skull width, and a decrease in skull length. These data were compared with skull phenotypes reported in other published reports on this model (Wu 2011) and correlated with skull phenotypes observed clinically in patients with NS and ACH. As shown in Table 25-3, daily treatment with 1600 μg / kg CNP prodrug or 500 μg / kg CNP improved this phenotype, resulting in a minor decrease in mean medial canthal distance but a significant increase in skull width and length. Similar to the femur and tibia, the CNP prodrug had a greater effect on skull length and width than CNP itself. Compared to WT mice treated with a mediator, WT mice treated with 1600 μg / kg CNP prodrug daily showed a significant increase in skull length only.

[0817] Table 25-3: WT mice and Raf1 mice treated for 6 weeks + / L613V Skull morphology of mice

[0818]

[0819] There was no test-related effect on the echocardiographic parameters, confirming that neither CNP nor CNP prodrug affects the function of normal or hypertrophic hearts.

[0820] In summary, under the same background, daily doses of 500 μg / kg CNP and 1600 μg / kg CNP were effective in WT mice (50% 129S1 / SvilmJ and 50% C57BL / 6J) and Raf1 mice. + / L613V Induces specific overgrowth in mice. Treatment with CNP induces Raf1 + / L613VThe skull phenotype of the mice was normalized, and to a greater extent, treatment with CNP prodrugs also restored the skull phenotype to normal. No fractures were observed in the animals clinically or by μCT imaging, but the treatment duration was prolonged. These animals were treated at 4 weeks of age instead of 3 weeks and had different strain backgrounds, which may have influenced these differences. Despite the presence of the hypertrophic phenotype, treatment with CNPs or CNP prodrugs did not have adverse cardiac effects.

[0821] Effects of CNP prodrugs on increased MAPK signaling at RIT1 levels

[0822] This study aims to evaluate the performance of Rit1 (Rit + / - The effects of activated M90I mutations on the growth of CNP and CNP prodrugs in mice were investigated. These effects contrast with those of the small molecule MEKi PD0235901.

[0823] Mutations in RIT1 associated with NS were less associated with short stature in NS mice compared to mutations in RAF1 (Yaoita 2016). M90I mutations in RIT1 increase signaling along the ERK1 / 2 MAPK pathway, which can be improved by CNP treatment. It has previously been shown that, compared to WT littermates, mice carrying the M90I mutation (Rit1) have a lower risk of developing short stature due to NS. + / - Heterozygous mice at 4 weeks of age have shorter body length and increased spleen and heart-to-body weight ratios (Castel 2019).

[0824] In this study, 30 WT and 45 8-week-old Rit1 mice were included. + / - Male mice were randomly divided into 10 groups. Animals were administered the carboxin or 500 μg / kg CNP daily via SC injection, or the carboxin or 3280 μg / kg CNP prodrug per dose via QAD. + / - The mouse control group received either daily intraperitoneal injections of the mediator or 5 mg / kg MEKi daily. All animals were treated for 10 weeks. The study design is shown in Table 25-4.

[0825] Table 25-4: In WT mice and Rit1 + / Design of pharmacodynamic studies in Noonan syndrome mice

[0826]

[0827]

[0828] Animals were weighed before each administration. Radiographic measurements of nose-to-anus length, tail length, long bones (tibia, femur, ulna, humerus), and vertebrae, as well as echocardiography, were collected at baseline, before the start of treatment, and 6 weeks after treatment. Radiographic measurements were also collected at week 10 of the study, before its termination. Animals were euthanized and necropsy was performed at week 10 of the study. Final body weight, nose-to-anus length, and tail length were measured, and the heart, lungs, liver, and spleen were weighed.

[0829] During the 10-week treatment period, WT mice treated with CNP and CNP prodrug showed significantly visible bone growth, and Rit... + / - Bone growth was also observed in mice, but the extent of growth was limited. The study was initially planned to be administered for 14 weeks, but was terminated at 10 weeks due to the observation of bone growth phenotypes and the absence of HCM phenotypes in animals treated with CNP and CNP prodrugs, rendering further administration ineffective.

[0830] With Raf1 + / L613V The same applies to mice, Rit + / - Mice did not exhibit the HCM phenotype, but their hearts were enlarged relative to their body weight, which was not significantly affected by CNP prodrugs or CNP treatment. Echocardiographic assessment showed that the test substance had no adverse effects on cardiac function.

[0831] For daily doses of 500 μg / kg CNP and 3280 μg / kg QAD CNP prodrug, the naso-anal length was significantly increased relative to the treatment group and the genotype-matched control group treated with the carcass. Figure 26 In WT animals, treatment with 3280 μg / kg QAD CNP prodrug resulted in a significantly longer nose-to-anus length compared to animals treated with CNP. + / - There was no difference in dose-response in mice. MEKi treatment also caused an increase in nose-anal length, but to a lesser extent than that achieved with CNP or CNP prodrug treatment. A similar trend was observed with respect to tail length.

[0832] Figure 27 This image shows representative images of WT mice treated with the causative agent and with CNP prodrug at baseline, 6 weeks of treatment, and 10 weeks of treatment. CNP prodrug treatment significantly increased long bone length and vertebral growth was evident. Early data from this study indicate a significant therapeutically relevant effect of CNP prodrug on bone growth, as the animals achieved the specific overgrowth phenotype previously observed in mice using CNP.

[0833] Pharmacodynamics of CNP prodrugs in NHP

[0834] Male stone crab macaques were treated with a CNP prodrug for one month to assess pharmacological activity, which was evaluated by radiographic and microscopic assessment of the skeleton and by assessment of biomarkers of bone formation. Pharmacokinetics and CV safety pharmacology were also evaluated. Animals were 2 years old and weighed 2.7 to 3.3 kg at the start of the study. The study design is shown in Table 25-5.

[0835] Table 25-5: Design of a 1-month pharmacological study in stone crab macaques

[0836]

[0837] On days 1, 8, 15, 22, and 29 of the study, the carrier drug or a CNP prodrug at a dose of 30 or 150 μg / kg (based on CNP content) was administered to animals weekly via SC injection. In the scapular region of the back, the dose was delivered alternately between the left side (days 1 and 15) and the right side (days 8 and 22). On day 29, the final dose was delivered to the untreated site in the midback.

[0838] Animals were monitored daily for death or visible evidence of toxicity, and detailed clinical observation and body weight measurements were performed weekly during the administration period. Food consumption was qualitatively monitored. Clinical chemistry and hematology samples were collected weekly and on the day of necropsy. Before treatment initiation, on study days 1, 8, 15, 22, and 29, and before necropsy on study day 31, biomarkers of mast chondrocyte differentiation (fragment of collagen X and the NC1 domain of collagen X [Pro-C10 HP]) in serum from fasting animals were measured; as well as bone metabolism / formation, i.e., type II collagen degradation (new type II collagen epitope [T2CM]); osteoblast activity (fragment of bone-specific alkaline phosphatase [BAP] and N-terminal type I collagen [Pro C1]); and type II collagen formation (fragment of N-terminal type IIB collagenogen [Pro C2]). Before treatment initiation and one and five days after each dose, on days 2, 6, 9, 13, 16, 20, 23, 27, and 30 of the study, the levels of type I collagen telopeptide (CTx-Ia; a biomarker of type I collagen degradation) and type II collagen telopeptide (CTx-II; a biomarker of type II collagen degradation), as well as cGMP (a biomarker of CNP cell activity), were measured in urine collected overnight from fasting animals. Plasma levels of atrial natriuretic peptide (ANP) were measured in PK samples collected after each dose to confirm that sustained supraphysiological levels of CNP did not interfere with ANP clearance via NPR-C.

[0839] Digital radiography of the femur / tibia / fibula, spine, and radius / ulna was performed under anesthesia in all animals before drug administration began on day 21 of the study and before necropsy on day 31. Qualitative evaluation of the radiographs was performed, and epiphyseal plate closure of the right proximal tibia / fibula and radius / ulna, as well as the right distal and proximal femur, was scored. Tibial and ulnar lengths (right and left) were obtained from the radiographs. Body and tail lengths of the anesthetized animals were measured directly using a flexible measuring tape during radiography.

[0840] On day 31 of the study, all animals were euthanized and subjected to full necropsy. The sternum, costochondral junction of the sixth rib, proximal left tibia, injection sites, and gross lesions were collected and examined under a microscope. Histological analysis of the left proximal tibial epiphyseal plate of each animal was performed to measure the thickness of the total epiphyseal growth plate and hyperplastic and hypertrophic areas.

[0841] All animals survived to scheduled necropsy. No clinical observations related to the assays or effects on body weight / weight gain, hematology, or clinical chemistry were observed. In animals treated with CNP prodrugs, plasma ANP levels were below the limit of quantitation (BLQ) at most time points, thus no definitive conclusions could be drawn regarding trends; however, the results indicate that continued delivery of CNP via CNP prodrug treatment does not induce competition for the common clearance receptor NPR-C.

[0842] On the second day of weekly dosing (days 9, 16, 23, and 30), the change in normalized cGMP relative to baseline increased in the treatment groups (groups 2 and 3) from the second dose onwards. The change in normalized cGMP relative to baseline was generally lower on day 6 of weekly dosing (days 13, 20, and 27) compared to the second day after weekly dosing (days 9, 16, 23, and 30). The increase in normalized cGMP was higher in group 3 (150 μg / kg weekly) compared to group 2 (30 μg / kg weekly), indicating that the pharmacological activity of CNP persisted in a dose-dependent manner over the 30 days of dosing.

[0843] No detectable therapeutic effect was observed on serum collagen X, Pro-C10 HP, T2CM, Pro-C1, or Pro C2. Changes in BAP concentrations relative to baseline were similar across all three groups, except for days 29 and 31 (the day of or two days after the fifth dose), at which point positive changes were observed in animals receiving weekly 150 μg / kg CNP prodrugs. Changes in urinary creatinine-normalized CTxIa concentrations relative to baseline were similar across all three groups, and no observable trend was noted. At most time points, urinary CTx-II concentrations were BLQ in all groups.

[0844] No significant treatment-related radiographic changes or worsening of previously observed radiographic findings were observed. No significant treatment-related changes were found in tibia, ulna, body, or tail length when compared to animals treated with cauterizers, possibly due to the shorter study duration.

[0845] No treatment-related gross findings were observed during necropsy, and all gross lesions were attributed to surgical implantation of the telemetry device. No CNP prodrug-related findings were observed at the injection site. As expected and previously observed with CNP treatment, a very low increase in cellular content in the hypertrophic zone of the epiphyseal plate was observed in animals treated weekly at 150 μg / kg (4 / 4), the proximal tibia (2 / 4), and the costochondrial junction of the sixth rib (1 / 4), indicating pharmacological activity in hypertrophic cartilage cells. A slight increase in the mean thickness of the hypertrophic zone of the proximal tibial epiphyseal plate was observed in animals treated weekly at 150 μg / kg (150 μg / kg). Although this change was not statistically significant, it was considered to be associated with the CNP prodrug and was microscopically correlated with increased cellular content in the hypertrophic zone in both animals at this dose. When compared with parallel control animals, there were no CNP prodrug-related changes in total epiphyseal growth plate thickness and hyperplasia thickness at 150 μg / kg per week, and no changes in any measured parameters at 30 μg / kg per week.

[0846] In summary, the CNP prodrug was well tolerated in male NHPs when administered at 30 or 150 μg / kg / week for a total of 5 doses over 1 month. Pharmacological activity (i.e., binding to NPR-B) was evident based on the increase in normalized urinary cGMP levels at both dose levels; however, effects on growth plate cell content and hypertrophic zone thickness were observed only in animals administered 150 μg / kg / week, suggesting that the 30 μg / kg / week dose level had a sub-therapeutic effect. During the short duration of this study, the effects on the growth plate did not translate into increases in bone, body, or tail length.

[0847] Similar to primary and secondary pharmacodynamics, the active component CNP in CNP prodrugs also provides information for the safety pharmacology analysis of CNP prodrugs. Safety pharmacology studies of CNP prodrugs focus on their effect on CV endpoints, due to changes in the pharmacokinetic profile of CNPs (i.e., C...). max (Reduce) the observed effects of CNP on heart rate and blood pressure. The following is a brief overview of the safety pharmacology assessment of CNP, followed by a detailed discussion of the safety pharmacology studies of CNP prodrugs.

[0848] CNP's safety pharmacology

[0849] No changes in CNP-related respiratory or central nervous system (CNS) parameters were observed after a single administration of 30, 100, or 300 μg / kg CNP to rats via SC. The no-effect level (NOEL) was not observed at 300 μg / kg in these studies.

[0850] In NHP, as expected based on the mechanism of action of CNP in vascular structures, direct administration of CNP induces vascular smooth muscle relaxation, followed by a dose-responsive decrease in blood pressure and a compensatory increase in heart rate. Overall, minimal or no changes in blood pressure / heart rate were observed in conscious NHP administered ≤10 μg / kg CNP. An increase in heart rate of approximately 25% was observed in conscious NHP administered 28 μg / kg CNP. In GLP CV safety pharmacology studies, no significant CV-related clinical signs were observed in conscious NHP administered 50 μg / kg CNP, while a decrease in blood pressure of <10% and an increase in heart rate of approximately 37% were observed. Significant CV-related clinical signs were observed in some conscious NHP administered ≥200 μg / kg CNP. These clinical signs consisted of the following: in the pilot-scale CV study, decreased activity was observed in 5 / 8 of the animals 40 to 60 minutes after administration on day 1, and in the primary phase 2 CV study, idiopathic, transient, and recurrent sternal recumbency or lateral decubitus position was observed in 3 / 8 of the animals during the first hour after administration. In the 28-day repeated-dose toxicity study, no significant CV-related clinical signs were observed in NHP treated with up to 300 μg / kg CNP. Differences between studies with significant CV-related observations may be related to the administration method (distal or manual) and the amount of time the technician was present in the room depending on the size of the animal cohort. A diminished effect on blood pressure reduction was observed after repeated daily administration of CNP, while there was no effect on heart rate increase, and this may be due to desensitization of NPR-B in vascular structures at higher repeated doses. A naïve-like response was observed after a clearance period of at least 2 days. It is possible that the severity of measurable and observable hemodynamic changes is generally limited because the distribution of NPR-B in vascular structures is primarily confined to peripheral vascular structures. Based on the absence of measurable changes in heart rate and blood pressure, and CV-related clinical observations, the noelary effect (NOEL) for subcutaneous CNP administration to conscious NHPs was 10 μg / kg in this study. No significant CV-related clinical observations were observed in conscious monkeys administered ≤50 μg / kg CNP via SC. QT interval prolongation was not observed in any of the studies.

[0851] Since no effects of CNP on CNS or lung function have been shown, there are no plans to conduct CNS or respiratory safety pharmacology studies on CNP prodrugs.

[0852] The aim is to reduce CNP C levels through continuous release of CNP prodrugs rather than daily pulse therapy. max This will improve the CV effect of CNP. This was confirmed in two studies conducted in stone crab macaques: a non-GLP study and a GLP-compliant study.

[0853] As described above, male stone crab macaques were administered a mediator weekly via SC injection or a CNP prodrug at a dose of 30 or 150 μg / kg weekly (based on CNP content), for a total of 5 doses. DSIPhysioTel was surgically implanted prior to the start of administration. ® Animals were remotely and continuously monitored for 24 hours using a digital M11 telemetry device, including heart rate, systemic arterial pressure (systolic, diastolic, pulse pressure, and mean arterial pressure), ECG parameters (PR, RR, QRS, QT interval, and corrected QT interval [QTc]), and body temperature. Monitoring was then repeated for 20 hours after each administration, starting 25 hours post-administration. A veterinary cardiologist also performed a qualitative assessment of the ECG at 36 hours post-administration. No effect on any CV parameters was observed. This study confirms that, as expected, repeated administration of pharmacologically active doses of CNP prodrugs does not cause a decrease in blood pressure or an increase in heart rate, which is typically observed with a single SC injection of CNP.

[0854] A GLP-compliant study was conducted, designed to evaluate the toxicity (described further below) of CNP prodrugs and formed CNPs in male and female stone crab macaques, as well as their pharmacological effects on the CV system.

[0855] In this study, male and female stone crab macaques were administered a mediator or a CNP prodrug (based on CNP content) via SC injection, with each dose administered at two-week intervals (days 1, 15, and 29 of the study) to avoid accumulation. The study design is shown in Table 25-6.

[0856] Table 25-6: Design of escalation-dose toxicity and CV safety pharmacology studies of CNP prodrugs in Lithocarpus macaques

[0857]

[0858] DSIPhysioTel was surgically implanted for up to 72 hours before the start of administration and subsequently on days 1, 15, and 29 of the study. ® The digital M11 telemetry device can remotely and continuously monitor an animal's heart rate, systemic arterial pressure (systolic pressure, diastolic pressure, pulse pressure, and mean arterial pressure), ECG parameters (PR, RR, QRS, QT interval, and QTc), and body temperature.

[0859] No CNP prodrug-related changes were observed in systemic blood pressure (mean arterial pressure, systolic and diastolic blood pressure, pulse pressure) and heart rate, as assessed up to 72 hours after administration of 150, 300, and 500 μg / kg. In female mice treated with the 500 μg / kg (third dose) CNP prodrug, a significant trend toward a decrease in mean heart rate measurements was observed, particularly pronounced during the dark cycle relative to the control, with some of these changes reaching statistical significance. However, individual heart rate measurements in female animals treated with the 500 μg / kg CNP prodrug remained comparable to their pre-dosage and / or pre-study values. This trend is partly due to additional individual variability caused by an increased heart rate measurement in a control animal in this case.

[0860] In summary, these studies confirm that the CV effect of CNP can be eliminated by reducing the steep peak of CNP exposure observed in daily delivery, while maintaining its pharmacological activity on endochondral bone growth.

[0861] Example 26: Pharmacokinetics of CNP Prodrugs

[0862] The pharmacokinetics of CNP have been thoroughly evaluated. Non-clinical PK characterization of the subcutaneously administered CNP prodrug was conducted in small animals (mice and rats) and large animals (NHP). These studies were conducted (or continued) alongside toxicity studies, which included three single-dose studies and two repeated-dose studies. Additionally, the study plans to characterize the metabolic stability, plasma protein binding, and drug-drug interaction potential of the CNP prodrug and its metabolites released after hydrolysis (lipid-linker; metabolite B).

[0863] Pharmacokinetics in mice

[0864] In a single-dose pharmacokinetic (PK) study conducted in 10-week-old male C57BL / 6J mice, CNP prodrugs were administered via a single SC injection of 500 μg / kg or 1600 μg / kg. Plasma concentrations were measured in each mouse at a single time point, with approximately three mice used for each time point. Non-compartmental analyses were performed using the mean plasma concentrations at each time point to determine PK parameters. Following SC injection, the CNP prodrug was slowly absorbed into the plasma, with the median T500 at the 500 μg / kg dose being [data missing]. max The median T value is 8 hours and 1600 μg / kg dose. max It is 4 hours. For both dose groups, the mean t½ for the CNP prodrug was approximately 10.5 hours. The dose-normalized area under the plasma concentration-time curve (AUC) and C... max Similar values ​​indicate that plasma exposure to the CNP prodrug increases proportionally with doses ranging from 500 to 1600 μg / kg. CNP is slowly released into plasma, with a mean T0.05...max The time to release was 4 hours at a dose of 500 μg / kg and 12 hours at a dose of 1600 μg / kg. Similar to the CNP prodrug, the CNP released from the CNP prodrug had a longer average time to release compared to the SC dose of CNP. 1 / 2 The duration of action was 9.6 hours for the 500 μg / kg dose and 9.9 hours for the 1600 μg / kg dose. Dose-normalized plasma concentrations were similar in both groups. Dose-normalized CNPAUC and C2O values ​​were similar in both dose groups. max Similarly, this indicates that plasma exposure to CNP increases proportionally with doses ranging from 500 to 1600 μg / kg. In C... max The percentage of downmetabolites (released CNP / CNP prodrug) was 0.07% at a dose of 500 μg / kg and 0.1% at a dose of 1600 μg / kg. Compared with the CNP prodrug, the percentage of metabolites representing the AUC of released CNP was 0.06% at a dose of 500 μg / kg and 0.08% at a dose of 1600 μg / kg.

[0865] Pharmacokinetics in rats

[0866] In a single-dose pharmacokinetic study conducted in adult rats, CNP prodrugs were administered at 560 μg / kg IV and at 150 μg / kg, 280 μg / kg, 560 μg / kg, or 1680 μg / kg SC. Following IV administration of the CNP prodrug, CNP was slowly released, reaching peak concentration (Tc) at 4 hours. max The average t of the released CNP 1 / 2 It is 10.8 hours, similar to CNP prodrug (8.9 hours). The area under the plasma concentration-time curve (AUC) from time 0 to infinity... 0-∞ The proportion of CNP metabolites released was 0.58%. The average t of CNPs released by male animals (10.7 hours) and female animals (10.9 hours) was... 1 / 2 Similarly. However, compared to females (45800 h*pM and 2380 pM, respectively), males released significantly higher levels of CNP (AUC and C). max The levels were slightly higher (60900 h*pM and 2800 pM, respectively). Following SC administration, the CNP prodrug was slowly absorbed into the plasma, with a median T value of [missing value] for all dose groups. max It is 8 hours. The average time for CNP prodrug is... 1 / 2The duration of exposure ranged from 6.72 hours at a 150 μg / kg SC dose to 10.5 hours at a 1680 μg / kg SC dose. A dose-proportional increase in CNP prodrug exposure was observed, with the mean dose-normalized exposure (AUC) of the CNP prodrug increasing for all SC doses from 150 μg / kg to 1680 μg / kg. 0-∞ The bioavailability of CNP prodrug SC doses was similar to that of Dose, and the dose-normalized plasma concentration curves actually overlapped. Bioavailability of CNP prodrug SC doses ranged from 17.67% to 21.71%. No consistent trend in bioavailability was observed between sexes. With slow release of CNP, the median CNP T... max Slightly delayed compared to CNP prodrug (T max (Approximately 10-12 hours after administration). The released CNP t 1 / 2 Similar to CNP prodrugs, the range is 7.6 to 12.8 hours. The mean exposure (AUC) of the released CNP... 0-∞ ) and C max The AUC increased with increasing dose from 150 μg / kg to 280 μg / kg and from 560 μg / kg to 1680 μg / kg, but was similar at 280 μg / kg and 560 μg / kg doses. Compared with the two high-dose groups (0.294% at 560 μg / kg and 0.323% at 1680 μg / kg), the AUC of the two low-dose groups was... 0-t The percentage of metabolites was high (0.461% at 150 μg / kg and 0.494% at 280 μg / kg). No consistent sex-based trend was observed in exposure to CNP prodrugs and released CNPs. max And AUC increased proportionally with increasing dose in the dose range of 280 μg / kg to 1680 μg / kg. For the released CNP, C max The AUC increased proportionally from doses of 150 μg / kg to 280 μg / kg and from 560 μg / kg to 1680 μg / kg. Pharmacokinetics in NHP

[0867] In repeated-dose studies conducted in NHP, CNP prodrugs (based on CNP content) were administered weekly via SC injection at 30 μg / kg or 150 μg / kg per week for one month. Following SC administration, the CNP prodrugs were slowly absorbed, with a median Tw at 30 μg / kg QW. max The median T was 24 hours after the first dose on day 1 of the study and 16 hours after multiple weekly doses on day 22 of the study, at a weekly dose of 150 μg / kg. maxThis refers to 48 hours after the first dose on day 1 of the study and 28 hours after multiple weekly doses on day 22 of the study. Compared with rodents, the CNP prodrug had a longer mean t... 1 / 2 The duration was 60 to 65.3 hours, which was consistent with the dose levels of 30 μg / kg QW and 150 μg / kg QW. At 168 hours after 3 weeks of administration at 30 μg / kg QW, the C-value of the CNP prodrug was observed. max AUC tlast AUC 0-∞ And trough plasma concentration (C 谷值 The cumulative effects were 2.09, 2.36, 3.37, and 3.15 times that of the CNP prodrug. After 3 weeks of administration at 150 μg / kg QW, CNP levels were observed at 168 hours. max AUC tlast AUC 0-∞ and C 谷值 The cumulative effects were 1.24 times, 1.5 times, 1.42 times, and 1.67 times. Apparent drug clearance on day 1 and day 22, apparent volume of distribution at terminal stage, and t... 1 / 2 The values ​​are similar, indicating that the CNP prodrug PK remains constant after repeated administration in NHP.

[0868] For most animals treated with 30 μg / kg QW on days 1 and 22 of the study, the plasma concentration of released CNPs was not quantifiable. To demonstrate the slow release of CNPs, the median T of the plasma concentration of released CNPs at 150 μg / kg QW was calculated. max The C-values ​​of released CNPs were observed at 8 hours and 48 hours after the first dose. Following three weekly doses of 150 μg / kg QW, C-values ​​of released CNPs were observed at 168 hours. max AUC 0-t AUC 0-∞ and C 谷值 The cumulative effects were 1.88 times, 2.62 t...

Claims

1. A pharmaceutical composition comprising a variant of C-type natriuretic peptide (CNP) PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO:1), a pharmaceutically acceptable excipient, and a carrier or diluent, wherein the CNP variant comprises an acid moiety, a spacer, a hydrolyzable linker, and is characterized by the structure shown in FIG11.

2. A pharmaceutical composition comprising (4R, 10S, 16S, 19S, 22S, 28S, 31S, 34S, 37S, 40S, 43S, 49S, 52R)-52-(2-((S)- ... -2-((S)-1-(L-prolyl-glycyl-L-glutamine-l-glutamine-L-histyl-pyrrolidine-2-carboxamido)-4-amino-4-oxobutamido)propamido)-5-guanidinylpentamido)-6-aminohexanoyl)-3-(4-hydroxyphenyl)propamido)-6-aminohexanoyl)acetamyl)propamido)-4-amino-4-oxobutamido)-6-aminohexanoyl)acetamyl)-6-aminohexanoyl)acetamyl)- 4-Methylpentamido)-3-hydroxypropamido)-6-aminohexano)acetami)-49-benzyl-28-((S)-sec-butyl)-34-(carboxymethyl)-40-((S)-33,51-dicarboxy-8-(2-hydroxyethyl)-6,12,21,30,35-pentoxo-14,17,23,26-tetraoxa-5,8,11,20,29,34-hexaazapentaylyl)-31-(3-guanidinopropyl)-16,22-bis( Hydroxymethyl)-10,37,43-triisobutyl-19-(2-(methylthio)ethyl)-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-hexadecoxo-1,2-dithia-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-hexaazacyclopentatriane-4-carboxylic acid, pharmaceutically acceptable excipients and carriers or diluents.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is lyophilized.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the carrier or diluent comprises a buffer.

5. The pharmaceutical composition of claim 4, wherein the buffer comprises a buffer selected from citrate, acetate, phosphate, TRIS, and combinations thereof.

6. The pharmaceutical composition of claim 5, wherein the buffer further comprises histidine, its salt, its solvate, or a solvate of a salt thereof.

7. The pharmaceutical composition according to any one of claims 4 to 6, wherein the buffer is present at a concentration of 5-15 mM.

8. The pharmaceutical composition according to any one of claims 4 to 7, wherein the pH is 3-9.

9. The pharmaceutical composition according to claim 8, wherein the pH is 4-6.

10. The pharmaceutical composition according to claim 9, wherein the pH is 5-6.

11. The pharmaceutical composition according to claim 10, wherein the pH is 5.2 or 5.

5.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the pharmaceutically acceptable excipient is selected from bulking agents, tension modifiers, antioxidants, surfactants, solubilizers, stabilizers, and combinations thereof.

13. The pharmaceutical composition according to claim 12, wherein the build-up agent is selected from mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24), and combinations thereof.

14. The pharmaceutical composition according to claim 12 or 13, wherein the accumulator comprises trehalose or a solvate thereof, mannitol or a combination thereof.

15. The pharmaceutical composition of claim 14, wherein the build-up agent comprises trehalose and mannitol in a weight ratio of 3:1 to 1:

1.

16. The pharmaceutical composition according to claim 15, wherein the weight ratio of trehalose to mannitol is 3.9:

1.

17. The pharmaceutical composition according to any one of claims 13 to 16, wherein trehalose is present in an amount of 3-6% by weight of the composition.

18. The pharmaceutical composition according to claim 17, wherein trehalose is present in an amount of 3.5-5.8% by weight of the composition.

19. The pharmaceutical composition according to claim 18, wherein trehalose is present in an amount of 3.8-4.8% by weight of the composition.

20. The pharmaceutical composition according to any one of claims 12 to 19, wherein the pharmaceutical composition comprises a tension modifier selected from sodium chloride, dextran, glucose, glycerol, sorbitol, xylitol, ethanol, and combinations thereof.

21. The pharmaceutical composition according to any one of claims 12 to 20, wherein the pharmaceutical composition comprises an antioxidant selected from methionine, ascorbic acid, salts of ascorbic acid, thioglycerol, and combinations thereof.

22. The pharmaceutical composition of claim 21, wherein the antioxidant is methionine.

23. The pharmaceutical composition according to any one of claims 1 to 22, wherein the pharmaceutical composition comprises a stabilizer or surfactant selected from glycine, sorbitol, polysorbate, and combinations thereof.

24. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition comprises polysorbate.

25. The pharmaceutical composition according to any one of claims 1 to 24, wherein the pharmaceutical composition comprises L-histidine, histidine monohydrochloride monohydrate, trehalose dihydrate, D-mannitol, L-methionine, and polysorbate.

26. The pharmaceutical composition according to any one of claims 1 to 24, wherein the pharmaceutical composition comprises a citrate buffer, trehalose dihydrate, D-mannitol, L-methionine, and polysorbate.

27. A pharmaceutical composition comprising: (4R, 10S, 16S, 19S, 22S, 28S, 31S, 34S, 37S, 40S, 43S, 49S, 52R)-52-(2-((S)- ...S)-2-((S)-2-(S)-2-((S)-2-(S)-2-(S)-2-(S)-2-(S)-2-(S)-2-(S)-2-(S)-2-(S)-2-(S)-2-(S)-2-(S)-2-(S)-2-(S)-2-(S)-2-(S)-2-(S)-2-(S)-2-(S)-2- )-1-(L-prolyl-glycyl-L-glutamine-l-glutamine-L-histyl)pyrrolidine-2-carbamoyl)-4-amino-4-oxobutamido)propamido)-5-guanidinopentamido)-6-aminohexanoyl)-3-(4-hydroxyphenyl)propamido)-6-aminohexanoyl)acetamyl)propamido)-4-amino-4-oxobutamido)-6-aminohexanoyl)-6-aminohexanoyl)acetamyl)-4-methylpentamido) )-3-hydroxypropamido)-6-aminohexamido)acetami)-49-benzyl-28-((S)-sec-butyl)-34-(carboxymethyl)-40-((S)-33,51-dicarboxy-8-(2-hydroxyethyl)-6,12,21,30,35-pentoxo-14,17,23,26-tetraoxa-5,8,11,20,29,34-hexaazapentadecyl)-31-(3-guanidinopropyl)-16,22-bis(hydroxymethyl)-10,37 ,43-Triisobutyl-19-(2-(methylthio)ethyl)-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-Hexadecoxo-1,2-dithia-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-Hexadecazacyclopentatriane-4-carboxylic acid, buffers containing histidine or its salts, and one or more pharmaceutically acceptable excipients.

28. The pharmaceutical composition of claim 27, wherein the buffer comprises L-histidine hydrochloride monohydrate.

29. The pharmaceutical composition according to claim 27 or 28, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient selected from: bulking agents, stabilizers, anti-adsorption agents, diluents, and combinations thereof.

30. The pharmaceutical composition according to any one of claims 27 to 29, wherein the pharmaceutically acceptable excipient comprises a bulking agent, the bulking agent comprising trehalose or a solvation thereof.

31. The pharmaceutical composition of claim 30, wherein the accumulator comprises trehalose dihydrate.

32. The pharmaceutical composition according to any one of claims 27 to 31, wherein the pharmaceutically acceptable excipient comprises a bulking agent, said bulking agent comprising mannitol.

33. The pharmaceutical composition according to claim 32, wherein the mannitol is D-mannitol.

34. The pharmaceutical composition according to any one of claims 27 to 33, wherein the pharmaceutically acceptable excipient comprises a stabilizer, said stabilizer comprising methionine.

35. The pharmaceutical composition according to claim 34, wherein the methionine is L-methionine.

36. The pharmaceutical composition according to any one of claims 27 to 35, wherein the pharmaceutically acceptable excipient comprises an anti-adsorption agent, and the anti-adsorption agent comprises polysorbate.

37. The pharmaceutical composition of claim 36, wherein the polysorbate is polysorbate 80.

38. The pharmaceutical composition according to any one of claims 27 to 37, wherein the pharmaceutical composition is substantially free of citrate buffer.

39. A pharmaceutical composition comprising: a C-type natriuretic peptide (CNP) variant, namely (4R, 10S, 16S, 19S, 22S, 28S, 31S, 34S, 37S, 40S, 43S, 49S, 52R)-52-(2-((S)-2-((S)-2-(2-((S)-2-((S)-2-((S)-2-((S)-2-(2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-1-(L-prolylglycyl-L-glutamineyl-L-glutamineyl-L-histyl)pyrrolidine-2-carboxamido)-4-amino- 4-O-Butyramido)propamido)-5-Guidinopentamido)-6-Aminohexamido)-3-(4-Hydroxyphenyl)propamido)-6-Aminohexamido)acetamamido)propamido)-4-Amino-4-O-Butyramido)-6-Aminohexamido)-6-Aminohexamido)acetamamido)-4-Methylpentamido)-3-Hydroxypropamido)-6-Aminohexamido)acetamamido)-4,9-Benzyl-28-((S) -sec-butyl)-34-(carboxymethyl)-40-((S)-33,51-dicarboxy-8-(2-hydroxyethyl)-6,12,21,30,35-pentoxo-14,17,23,26-tetraoxa-5,8,11,20,29,34-hexaazapentadecyl)-31-(3-guanidinopropyl)-16,22-bis(hydroxymethyl)-10,37,43-triisobutyl-19-(2-(methylthio)ethyl)-6, 9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-hexadecoxo-1,2-dithia-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-hexaazacyclopentatriane-4-carboxylic acid; histidine buffer; trehalose dihydrate; D-mannitol; L-methionine; polysorbate 80; and water.

40. The pharmaceutical composition of claim 39, wherein the CNP variant is present at a concentration of 10 mg / mL.

41. The pharmaceutical composition of claim 39, wherein the CNP variant is present at a concentration of 30 mg / mL.

42. The pharmaceutical composition according to any one of claims 39 to 41, wherein the histidine buffer comprises L-histidine and histidine monohydrochloride monohydrate.

43. The pharmaceutical composition according to claim 42, wherein L-histidine is present at a concentration of 0.35 mg / mL.

44. The pharmaceutical composition according to claim 42, wherein L-histidine is present at a concentration of 2.2 mM.

45. The pharmaceutical composition according to any one of claims 42 to 44, wherein L-histidine monohydrochloride monohydrate is present at a concentration of 1.6 mg / mL.

46. ​​The pharmaceutical composition according to any one of claims 42 to 44, wherein L-histidine monohydrochloride monohydrate is present at a concentration of 7.8 mM.

47. The pharmaceutical composition according to any one of claims 39 to 46, wherein the trehalose dihydrate is present at a concentration of 58 mg / mL.

48. The pharmaceutical composition according to any one of claims 39 to 46, wherein the trehalose dihydrate is present at a concentration of about 127 mM to about 153 mM.

49. The pharmaceutical composition according to claim 48, wherein the trehalose dihydrate is present at a concentration of 127 mM.

50. The pharmaceutical composition according to claim 48, wherein the trehalose dihydrate is present at a concentration of 153.3 mM.

51. The pharmaceutical composition according to any one of claims 39 to 50, wherein the D-mannitol is present at a concentration of 15 mg / mL.

52. The pharmaceutical composition according to any one of claims 39 to 50, wherein the D-mannitol is present at a concentration of about 68 mM to about 82 mM.

53. The pharmaceutical composition according to claim 52, wherein the D-mannitol is present at a concentration of 68.1 mM.

54. The pharmaceutical composition according to claim 52, wherein the D-mannitol is present at a concentration of 82.3 mM.

55. The pharmaceutical composition according to any one of claims 39 to 54, wherein the L-methionine is present at a concentration of 0.7 mg / mL.

56. The pharmaceutical composition according to any one of claims 39 to 54, wherein the L-methionine is present at a concentration of 4.9 mM.

57. The pharmaceutical composition according to any one of claims 39 to 56, wherein the polysorbate 80 is present at a concentration of 0.05 mg / mL.

58. The pharmaceutical composition according to any one of claims 39 to 56, wherein the polysorbate 80 is present at a concentration of 0.005% (v / v).

59. The pharmaceutical composition according to any one of claims 1 to 58, wherein the composition exhibits a lower Cmax and a higher AUC compared to the free drug.

60. A pharmaceutical kit comprising a pharmaceutical composition according to any one of claims 1 to 58.

61. A pharmaceutical kit comprising a pharmaceutical composition according to any one of claims 39 to 58, wherein the CNP variant is present in an amount of 13 mg.

62. A pharmaceutical kit comprising a pharmaceutical composition according to any one of claims 39 to 58, wherein the CNP variant is present in an amount of 39 mg.

63. A method for treating a subject with bone-related conditions or skeletal dysplasia, the method comprising administering to the subject the composition according to any one of claims 1 to 59.

64. The method according to claim 63, wherein the bone-related condition or skeletal dysplasia is selected from the group consisting of: osteoarthritis, hypophosphatemic rickets, achondroplasia, decreased cartilage production, short stature, dwarfism, osteochondrodysplasia, lethal achondroplasia, osteogenesis imperfecta, chondrodysplasia punctate chondrodysplasia, homozygous achondroplasia, brachydactyly, congenital lethal hypophosphatase syndrome, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, pedicled punctate chondrodysplasia, Janssen type metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, skeletal dysplasia, malformation dysplasia, etc. Congenital short femur, Langer type mid-limb dysplasia, Nivig type mid-limb dysplasia, Robinnoc syndrome, Reinhardt syndrome, acrodysplasia, peripheral bone dysplasia, Knifell's dysplasia, fibrocartilage hyperplasia, Roberts syndrome, acromegaly, microlimb syndrome, Moquer syndrome, Knifell's syndrome, variability dysplasia and vertebral epiphyseal dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Lerreville's disease), PTPN11 mutation (Noonan syndrome), insulin-like growth factor 1 receptor (IGF1R) mutation, osteoporosis, and idiopathic short stature.

65. A method for elongating bones or increasing long bone growth in a subject in need, the method comprising administering to the subject a composition according to any one of claims 1 to 59, wherein the administration causes bone elongation or increases long bone growth.

66. The method according to any one of claims 63 to 64, wherein the composition is administered subcutaneously, intradermally, intra-articularly, orally, or intramuscularly.

67. The method according to any one of claims 63 to 66, wherein the composition provides a prolonged release composition.

68. The method according to any one of claims 63 to 66, wherein the composition is applied once every 5 days, once a week, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months.

69. A method for treating CNP-responsive conditions or symptoms, the method comprising: -Administer the composition according to any one of claims 1 to 58 to the subject, and - Monitor the levels of at least one bone-related or cartilage-related biomarker in the subjects. An increase in the level of at least one bone-related or cartilage-related biomarker indicates that the CNP variant has a therapeutic effect on the subject or the condition or symptom.

70. The method of claim 69, further comprising adjusting the amount or frequency of application of the composition, wherein... i) If the level of at least one bone-related or cartilage-related biomarker is below the target level, increase the dosage or frequency of the composition; or ii) If the level of at least one bone-related or cartilage-related biomarker is higher than the target level, reduce the amount or frequency of application of the composition.

71. The method according to claim 69 or 70, wherein the at least one bone-related or cartilage-related biomarker is selected from the group consisting of: CNP, cGMP, type II collagen propeptide and fragment thereof, type II collagen and fragment thereof, type I collagen C-terminal peptide (CTx), osteocalcitonin, proliferating cell nuclear antigen (PCNA), type I collagen precursor propeptide (PINP) and fragment thereof, type I collagen and fragment thereof, chondroitin sulfate, collagen X, alkaline phosphatase, proliferating cell nuclear antigen (PCNA), type I collagen precursor propeptide and fragment thereof. Fragments, type I collagen and its fragments, agglutinin-glycan chondroitin sulfate, collagen X, CXM (non-collagen 1 (NC1) domain of type X collagen), NTproCNP and alkaline phosphatase, N-terminal type I collagen propeptide, bone-specific alkaline phosphatase, N-terminal propeptide of type I collagen / pro-N-propeptide of type I collagen (PINP), cross-linked type I collagen C-terminal peptide (CTx), cross-linked type I collagen N-terminal peptide (NTx) tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomic readouts and CNP-variant bioactivity.

72. The method according to any one of claims 63 to 71, wherein the administration increases the annualized growth rate (AGV) of the subject at 12 months, optionally compared to baseline or normal control.

73. The method of claim 72, wherein the subject's AGV increases over a period of one year or two years or longer.

74. The method according to any one of claims 63 to 73, wherein the administration improves the height Z-score at 12 months, optionally compared to baseline or normal control.

75. The method according to any one of claims 63 to 74, wherein the subject is older than 3 years.

76. The method according to any one of claims 63 to 75, wherein the subject is between 3 and 17 years old.

77. The method according to any one of claims 63 to 76, wherein the subject has an open epiphysis.

78. The method according to any one of claims 63 to 77, wherein the composition is administered at a dose of about 5 μg / kg to 500 μg / kg or about 15 μg / kg to 350 μg / kg.

79. The method according to any one of claims 63 to 77, wherein the administration does not cause cardiovascular (CV) side effects.

80. The method of claim 79, wherein the CV side effects are changes in systemic blood pressure, mean arterial pressure, systolic and / or diastolic blood pressure, pulse pressure, or heart rate.

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